Evaluation of cryopreserved ram semen following
fertilization in vitro
by
MASILO HENOKE MOHLOMI
Submitted in partial fulfilment of the requirements
for the degree
Master of Science in Agriculture
in the
Department Animal, Wildlife and Grassland Sciences
Faculty of Natural and Agricultural Sciences
University of the Free State
Bloemfontein
Supervisor: Mr. M. B. Raito
Co-Supervisors: Prof. J.P.C. Greyling
Dr. A.M. Jooste
July 2015
DEDICATION
To my mother ('Mamasilo Mohlomi), and my sisters ('Mampoeakae,'Mat'soloane, 'Malikhoa
and Puleng Mohlomi) and my brothers ('Mutsi and Sello Mohlomi) for their support.
i ACKNOWLEDGEMENTS
The author wish to acknowledge the following:

God for providing me with life and strength to come up to this far with this work.

My supervisor Mr M.B. Raito and co-supervisors Dr. A.M. Jooste and Prof. J.P.C.
Greyling for their excellent supervision.

Mamasilo Mohlomi, Mampoeakae Mohlomi, Matsoloane Mohlomi, The National
Manpower Development Secretariat (Lesotho) and Prof. J.P.C. Greyling for their
financial support.

Dr. M.D. Fair for the assistance with the statistical analyses of the data.

S. Kazetu, M. Kuena and K. Long for their practical assistance rendered during this
study.

Mrs H. Linde for assistance in compiling and printing of this thesis.
ii DECLARATION
I hereby declare that this dissertation submitted by me to the University of the Free State for
the degree, Master of Science in Agriculture, is my own independent work and has not
previously been submitted by me at another University. I furthermore cede copyright of the
dissertation in favour of the University of the Free State.
Masilo Henoke Mohlomi
Bloemfontein
July, 2015
iii TABLE OF CONTENTS
Page
Dedication
i
Acknowledgements
ii
Declaration
iii
Table of contents
iv
List of Tables
ix
List of Figures
x
List of Plates
xi
List of Abbreviations
xii
CHAPTER 1
1
GENERAL INTRODUCTION
1
CHAPTER 2
5
LITERATURE REVIEW
5
2.1
Male reproductive anatomy
5
2.1.1
Spermatogenesis
6
2.1.2
The structure of the sperm cell
8
2.1.3
The seminal vesicle
9
2.1.4
The prostate gland
10
2.1.5
Semen
10
2.2
Factors affecting the quality of the sperm
11
2.2.1
11
Environment
iv 2.3
2.2.2
Season of the year
11
2.2.3
Age of the male
12
2.2.4
Nutrition
12
The collection of the ram semen
13
2.3.1
The artificial vagina
13
2.3.2
Electrical stimulation
14
2.3.3
The principle involved in the use of the artificial vagina and electro-
2.3.4
2.4
ejaculator
14
The hormonal principles
16
Evaluation of the semen
17
2.4.1
17
Semen volume
2.4.2 Colour and density of the semen
18
2.4.3
pH
18
2.4.4
Sperm motility and percentage live sperm
18
2.4.5
Semen smears
19
2.5
Dilution of semen
19
2.6
Storage of the semen
20
2.6.1
Liquid semen
20
2.6.2
Frozen semen
21
2.6.2.1 Semen freezing procedures
23
Cryoprotective agents
24
2.6.3
2.7
In vitro fertilization
25
2.8
The fertilization process
26
v 2.9
Sperm capacitation
27
2.10
Factors affecting oocyte quality
31
2.10.1 Maturation process and media used
31
2.10.2 Age of the donor
32
2.10.3 Ovarian follicular size
32
2.10.4 Body condition and nutritional status
32
2.10.5 Morphology of the ovaries
33
2.10.6 Environmental factors and IVEP
33
2.10.7 Reproductive status of the animal and oocyte competence
33
CHAPTER 3
35
MATERIALS AND METHODS
35
3.1
Study area
35
3.2
Experimental animals
35
3.2.1
Dorper rams
35
3.2.2
South African Mutton Merino rams
36
3.3
Management of experimental animals
37
3.4
Preparation of the semen extenders
40
3.5
Semen collection and quality evaluation
40
3.5.1
Semen evaluation
43
3.5.1.1 Colour of the ejaculate
43
3.5.1.2 Semen volume
43
3.5.1.3 Semen wave motion
43
vi 3.5.1.4 Sperm concentration (semen density)
44
3.5.1.5 Sperm viability and morphology
44
3.6
Cryopreservation of ram semen
45
3.7
Thawing of semen for the post-thaw semen analysis
48
3.8
In vitro fertilization
49
3.8.1
Ovary collection
49
3.8.2
Method of oocytes collection
49
3.8.3
Oocytes maturation in vitro
49
3.8.4
In Vitro fertilization (IVF)
50
3.8.5
In Vitro embryo culture
50
3.9
Data Analysis
51
CHAPTER 4
52
RESULTS
52
4.1
Effect of breed (Dorper and SAMM) on fresh semen before
cryopreservation
4.2
52
Effect of breed, different extenders and different freezing protocols
on the viability of frozen ram semen for different incubation time periods
after thawing
4.3
52
Effect of breed, extender and freezing protocol on the in vitro
performance of thawed ram semen
54
CHAPER 5
57
DISCUSSION
57
vii 5.1
5.2
Effect of breed (Dorper and SAMM) on fresh semen parameters
prior to cryopreservation
57
5.1.1
Semen volume
57
5.1.2
Semen colour
58
5.1.3
Sperm motility
58
5.1.4
Sperm concentration
58
5.1.5
Sperm viability
59
5.1.6
Sperm morphology
59
Effect of breed difference, different extenders and different freezing
protocols on the viability of frozen-thawed ram semen
5.3
59
Effect of breed, extender and freezing protocol on the in vitro performance
of thawed ram semen
61
CHAPTER 6
63
GENERAL CONCLUSIONS
63
RECOMMENDATIONS
64
ABSTRACT
65
REFERENCES
69
viii LIST OF TABLES
Page
Table 2.1
Relations of semen colour and number of sperm cells (density) per
ejaculate in the ram
Table 2.2
18
Relationship between sperm motility and the percentage live sperm
in the ram
19
Table 4.1
Effect of breed on fresh ram semen quality
52
Table 4.2
Mean (±SE) post-thaw sperm motility of Dorper ram semen frozen
using different extenders and freezing protocols at different incubation
intervals following thawing
Table 4.3
53
Mean (±SE) post-thaw sperm motility of SAMM ram semen frozen
using different extenders and freezing protocols at different incubation
intervals following thawing
Table 4.4
54
Mean (±SE) effect of extender and freezing protocol on in vitro
fertilization of ram semen
55
ix LIST OF FIGURES
Page
Figure 2.1
Reproductive tract of a ram
5
Figure 2.2
Sequence of events and time involved during spermatogenesis
7
Figure 4.1
Effect of breed on in vitro fertilization results of frozen-thawed ram
semen
55
x LIST OF PLATES
Page
Plate 3.1
Dorper ram used as a semen donor
36
Plate 3.2
South African Mutton Merino ram used as a semen donor
37
Plate 3.3
Cleaning process to ensure a healthy environment for the rams
38
Plate 3.4
Water trough placed in each pen
39
Plate 3.5
Preparation of extenders for semen cryopreservation
40
Plate 3.6
Restraining a teaser ewe inside the semen collection pen
41
Plate 3.7
Preparation of artificial vagina before semen collection
42
Plate 3.8
Collection of semen from the rams using the artificial vagina
43
Plate 3.9
Haemocytometer used for sperm counting
44
Plate 3.10
Flask for transportation of semen from collection area to the freezing
Laboratory
Plate 3.11
46
Semen straws loaded into the programmable freezer for
cryopreservation
Plate 3.12
47
Semen straws suspended 3 – 4 cm above liquid nitrogen vapour for
cryopreservation
Plate 3.13
47
Liquid nitrogen tank used for the storage of semen after
cryopreservation
48
xi LIST OF ABBREVIATIONS
e.g
for example
min
minutes
hr
hour
˚C
Degree Celcius
µm
Micron
%
percentage
µl
Microliter
/
or
ml
milliliter
mg
milligram
mm
millimeter
mm2
millimeter square
AV
Artificial Vagina
IVF
In Vitro Fertilization
COC’s
Cumulus Oocyte Complexes
mPBS
modified Phosphate Buffered Saline
BSA
Bovine Serum Albumin
BCB
Brilliant Cresyl Blue
FBS
Fetal Bovine Serum
SAMM
South African Mutton Merino
BO
Bracket and Oliphant
SOF
Synthetic Oviduct Fluid
xii kg
Kilogram
LN2
Liquid Nitrogen
et al.
And others
SAS
Statistical Analysis System
ATP
Adenosine Triphosphate
DNA
Dioxynucliec Acid
GnRH
Gonadotropin-releasing Hormone
LH
Luteinizing Hormone
FSH
Follicle Stimulating Hormone
CO2
Carbon dioxide
ARTs
Assisted Reproductive Technologies
pH
Potential hydrogen
OPU
Ovum pick-up
MOET
Multiple Ovulation and Embryo Transfer
IVEP
In vitro Embryo Production
xiii CHAPTER 1
GENERAL INTRODUCTION
Reproductive success is a key component of economic production in ruminants, affecting both
animal productivity and genetic progress (Bauersachs et al., 2010). The reproduction success
in any animal production enterprise is then of high economical importance and this can be
achieved with better applied knowledge of reproduction physiology and application of certain
reproductive technologies.
When considering reproductive technologies as such, artificial insemination (AI) is probably
one of the most important reproductive techniques to accelerate the genetic improvement of
animals. The widespread use of AI in cattle has ultimately allowed accurate genetic evaluation
and rapid dissemination of genetic merit on a national and international basis to the benefit of
both the animal breeder and the consumer. It has also enabled the use of sophisticated data
analysis procedures to be implemented to identify animals with superior performance.
The availability of an efficient sheep AI service would also yield similar benefits, and would
greatly enhance the scope for pedigree and commercial breeders to respond positively and
effectively to consumer demands. The widespread use of AI and the realization of its full
potential then depend essentially on the use of frozen semen and on the availability of
techniques that could result in acceptable fertility. However, the poor fertility obtained when
frozen-thawed ram semen is used for cervical insemination in sheep has stimulated widespread
research interest in the sheep industry. Gil et al., (2003) stated that the relatively low success
rate of cervical AI with frozen semen in sheep has limited a wider application of the technique,
calling for an improvement of insemination technique itself and/or of the survival rate of the
frozen-thawed sperm. The short life span of fresh semen has on the other hand been reported
to be a constraint in the use of AI in genetic improvement programs for sheep (O’Hara et al.,
2010).
The alternative is laparoscopic AI as an effective method of insemination when using frozenthawed semen to extend the life span, but the procedure is expensive, thus limiting its use.
Welfare concerns may also limit the use of the laparoscopic AI procedure. It is generally
1 desirable and necessary to develop non-surgical procedures that could form the basis of making
AI a practical reality in the sheep industry.
Apart from AI, facilitation of genetic improvement of animals is done through the use of certain
other several assisted reproductive technologies (ART’s) such as semen and embryo
cryopreservation, estrous synchronization, multiple ovulation and embryo transfer (MOET), as
well as in vitro embryo production (IVEP). The cryopreservation of bovine semen and embryos
has made great progress in recent years, but little progress has been obtained in the small stock
industry (Zhu et al., 2001). Cryopreservation of gametes is seen as an important technique for
long time storage of semen and embryos for future use in the dissemination of superior genetic
material. The long term conservation of sperm is especially crucial for in vitro fertilization
(IVF) and/or AI purposes (Merlo et al., 2008). Cryopreservation ultimately creates the
opportunity to maintain superior genetic material at low costs, and also conserve endangered
species or breeds (Gonzalez-Bulnes et al., 2004; Mapletoft and Helser, 2005). As well as
providing some security with respect to disasters or outbreaks of disease that may seriously
affect animal population survival (Kirkwood and Colenbrander, 2001). However, the
cryopreservation process exposes sperm cells to physical and chemical stress and less than 50%
of the sperm cells may survive, with fertilizing ability being maintained (Waterhouse et al.,
2006).
It has further been recorded that some cryopreservation techniques, such as slow freezing are
expensive, may cause physical damage to the sperm and embryos, due to crystal formation and
are time consuming (Naik et al., 2005). The vitrification technique on the other hand may
cause damage due to cryoprotectant toxicity (Naik et al., 2005; Sharma et al., 2006). Slow
cooling however, seems to be the most important element used in the preservation technique
in sheep, when compared to vitrification (Thuwanut, 2007).
Tests that set minimum standards for semen used for artificial insemination (Mocé and
Graham, 2008) have limited value for predicting subsequent fertility of the semen sample
(Mocé and Graham, 2008). However, it is documented that semen evaluation techniques such
as sperm binding, oocyte penetration and in vitro fertilization estimates functional aspects of
spermatozoa (Flowers et al., 2009). The assessment of functional sperm parameters under
capacitating conditions has been proposed (Petrunkina et al., 2007). In contrast, the type of
extender affects fertilization potential while it has no effect on developmental potential up to
the blastocyst stage (Forouzanfar et al., 2010). Indeed, extender has a major effect on post2 thawed semen viability (Paulenz et al., 2002; Valente et al., 2010). In addition, semen quality
is negatively affected by freezing procedures (Nur et al., 2010) and thus, experiments to
examine the outcomes of the use of different freezing protocols have been proposed (Ramón
et al., 2013).
In vitro embryo production (IVEP) was first performed in order to produce relatively cheap
embryos on a large scale for various experimental procedures (Wani, 2002). Besides for
experimental purposes, IVEP can be seen as a possible method to produce embryos in
abundance to improve the reproductive efficiency of livestock (Rust and Visser, 2001). Early
stage embryos are required for the production of clones, transgenics, sexed embryos and for
the diagnosis of genetic defects (Wani, 2002). In vitro embryo production and fertilization are
now important technologies for obtaining live offspring (Kikuchi et al., 2009). For a viable
embryo to be produced, good quality sperm and oocytes are needed. The quality of oocytes is
of major importance in assuring the developmental competence of embryos, which is more
apparent and is determined by the oocyte’s nuclear and cytoplasmic maturation attained during
growth in the follicle (Sirard, 2001). Oocytes from small follicles (2-3 mm) have been shown
to have a reduced developmental in vitro competence due to lack of pre-maturation factors that
should occur during the final follicular growth phase (Cognie et al., 2004). A competent oocyte
is generally described as the oocyte which is able to sustain embryonic development to term
(Brevini-Gandolfi and Gandolfi, 2001). The number of high quality oocytes harvested from an
ovary is an important consideration in the in vitro production of embryos. Oocytes for in vitro
fertilization are generally collected from one of the following sources: the oviducts soon after
ovulation, mature follicles shortly before ovulation or immature and atretic follicles, usually
from abattoir material (Wani, 2002).
Ovaries from slaughtered animals are then the cheapest and most abundant source of primary
oocytes for large scale production of embryos through IVEP (Wani, 2002). To date, some
research has been done on IVEP in sheep (Wani et al., 2000; Galli et al., 2001; Wani, 2002,
Rao et al., 2002; Cognie et al., 2003; Katska-Ksiazkiewicz et al., 2004; Locatelli et al., 2006;
Cox and Alfaro, 2007 and Cocero et al., 2011). As research has also been done on breed effect
on semen and cryopreservation (Mahoete, 2010; Maghaddam et al., 2012).
Estrous synchronization as one of the available assisted reproductive technologies, can
contribute to some extent to the improvement of farm animal productivity. This involves the
application of the knowledge of animal’s hormonal activity to manipulate the sexual cycle.
3 Effective estrous synchronization can then facilitate the use of timed AI (Sa Filho et al., 2009).
Progress in the field of estrous synchronization has been directly linked to the discovery of a
wave-like pattern of follicular development (Day et al., 2010). Various relationships between
fertility and aspects of follicular development, such as follicle size, length of the pro-estrus
phase, follicular estradiol production and progesterone concentrations during follicular
development to fertility have emerged and led to fundamental investigations into the
mechanisms underlying these aspects (Day et al., 2010).
Multiple ovulation and embryo transfer (MOET) is a potential technique for increasing the
efficiency of breeding program in domestic animal production. Hafez and Hafez (2000)
documented that embryo transfer can be used to rapidly increase rare bloodlines, to obtain more
offspring from valuable females and to accelerate genetic progress by facilitating progeny
testing in females and thus reducing the generation interval.
In the sheep production industry, there is still much work to be done to improve reproductive
efficiency.
The objectives of the present study were thus as follows:
A. Evaluate the effect of breed on fresh ram semen quality parameters.
B. Compare the sperm motility of ram semen from two sheep breeds following
cryopreservation and thawing, based on 4 time intervals (0, 30, 60 and 120 minutes
after thawing).
C. Compare the effect of two extenders on ram semen cryopreservation of two breeds of
sheep.
D. Compare the effect of two ram semen freezing protocols of two sheep breeds
cryopreserved using two extenders.
E. Test the fertilizing ability of the frozen-thawed ram semen following cryopreservation.
4 CHAPTER 2
LITERATURE REVIEW
In this chapter the different phases involved with processes of semen collection,
cryopreservation and in vitro fertilization in sheep are reviewed in detail. Before discussing
these phases a look at the male reproductive anatomy, spermatogenesis and factors affecting
semen quality will be made.
2.1 Male reproductive anatomy
The semen production processes (spermatogenesis) takes place in the ram reproductive tract
which has the following physiological anatomy as shown in the figure below.
Figure 2.1 Reproductive tract of a ram (Simmons & Ekarius, 2001)
The testis is composed of coiled seminiferous tubules, in which the sperm are formed (Ganong,
2011), after which the sperm cells are then transported to the epididymis. The epididymis is
5 then the primary location for the maturation and storage of sperm prior to ejaculation
(Costanzo, 2006; Berne and Levy, 2008). The epididymis leads into the vas deferens, which
enlarges into the ampulla of the vas deferens immediately before the vas deferens enters the
body of the prostate gland. The two seminal vesicles, one located on each side of the prostate
gland, then empty the seminal fluid into the prostatic end of the ampulla, and the contents from
both the ampulla and the seminal vesicles pass into an ejaculatory duct leading through the
body of the prostate gland and then emptying into the internal urethra. Prostatic ducts also
empty fluid from the prostate gland into the ejaculatory duct and from there into the prostatic
urethra (Guyton and Hall, 2010; Ganong, 2011).
The urethra is the last connecting link from the testis to the exterior. The urethra being supplied
with mucus derived from a large number of minute urethral glands located along its entire
extent and even more so from bilateral bulbo-urethral glands (Cowper's glands), located near
the origin of the urethra.
2.1.1 Spermatogenesis
During the formation of the embryo, the primordial germ cells migrate into the testes and
become immature germ cells called spermatogonia which lie in two or three layers of the inner
surfaces of the seminiferous tubules. The spermatogonia then begin to undergo mitotic
divisions, beginning at puberty, and continually proliferate and differentiate through definite
stages of development, to eventually form sperm cells (Ganong, 2011). Events and time
involved during spermatogenesis are shown on the figure below.
Spermatogenesis occurs in the seminiferous tubules during the active sexual life of the male as
the result of stimulation by anterior pituitary gonadotrophic hormones. In the first stage of
spermatogenesis, the spermatogonia migrate between the Sertoli cells, towards the central
lumen of the seminiferous tubules. The Sertoli cells are very large, with overflowing
cytoplasmic envelopes that surround the developing spermatogonia all the way to the central
lumen of the seminiferous tubule (Ganong, 2011). Spermatogonia that cross the barrier into the
Sertoli cell layer become progressively modified and enlarged to form large primary
spermatocytes. Each of these, in turn, undergoes meiotic division to form two secondary
spermatocytes. After another few days, these too divide to form spermatids which are
eventually modified to become spermatozoa (sperms) (Cheng and Mruk, 2010).
6 Figure 2.2 Sequence of events and time involved during spermatogenesis (Cheng and
Mruk, 2010)
During the change from the spermatocyte stage to the spermatid stage, the 46 chromosomes
(23 pairs of chromosomes) of the spermatocyte are divided. Thus 23 chromosomes go to one
spermatid and the other 23 to the second spermatid (Ganong, 2011). In each spermatogonium,
one of the 23 pairs of chromosomes carries the genetic information that determines the sex of
each individual offspring. The pair being composed of one X chromosome, is called the female
chromosome, and one with a Y chromosome, the male chromosome. During meiotic division,
the male Y chromosome goes to one spermatid that then becomes a male sperm, and the female
X chromosome goes to another spermatid that becomes a female sperm (Guyton and Hall,
2006).
7 2.1.2 The structure of the sperm cell
When the spermatids are first formed, they still have the usual characteristics of epithelioid
cells, but soon begin to differentiate and elongate into spermatozoa. The spermatozoa are
broadly composed of a head and a tail. The head is comprised of the condensed nucleus with
only a thin cytoplasmic and cell membrane layer around its surface (Guyton and Hall, 2006).
On the outside anterior a two thirds of the head is a thick cap, called the acrosome that is formed
mainly from the Golgi apparatus. This acrosome contains a number of enzymes, similar to
those found in the lysosomes of a typical cell, including hyaluronidase (which can digest
proteoglycan filaments of tissues) and powerful proteolytic enzymes (which can digest
proteins). These enzymes then play important roles in allowing the sperm to enter the ovum
and fertilize it (Guyton and Hall, 2006).
According to Guyton and Hall ( 2010), the tail of the sperm, called the flagellum, has three
major components mentioned below:
(1) a central skeleton constructed of 11 microtubules, collectively called the axoneme, similar
in structure to that of cilia found on the surfaces of other types of cells
(2) a thin cell membrane covering the axoneme; and
(3) a collection of mitochondria surrounding the axoneme in the proximal portion of the tail
(called the body of the tail).
Back and forth movement of the sperm tail (flagella movement) provides motility to the sperm
cells. This movement results from a rhythmical longitudinal sliding motion between the
anterior and posterior tubules that make up the axoneme. The energy for this process is supplied
in the form of adenosine triphosphate (ATP), synthesized by the mitochondria in the body of
the tail.
After formation in the seminiferous tubules, the sperm require several days to pass through the
tubule of the epididymis. Sperm removed from the seminiferous tubules and from the early
portions of the epididymis are non-motile, and they cannot fertilize an ovum. However, after
the sperm have been in the epididymis for some time, they develop the capability of motility
(Ganong, 2011), even though several inhibitory proteins in the epididymal fluid still prevent
final motility until after ejaculation. Only a small quantity of sperms cells can be stored in the
epididymis, most are stored in the vas deferens. During this time, they are kept in a deeply
8 suppressed inactive state by multiple inhibitory substances in the secretions of the ducts.
Conversely, with a high level of sexual activity and ejaculations.
During semen
cryopreservation, at temperatures below -80°C, the highly concentrated, viscous solution
within and outside the sperm turns into a relatively stable glassy matrix, which is basically
maintained when sperm are stored at -196°C (LN2) (Rodrequize-Martines et al., 2009).
After ejaculation, the sperm cell become motile, and also becomes capable of fertilizing the
ovum, a process called maturation. The Sertoli cells and the epithelium of the epididymis
secrete a special nutrient fluid that is ejaculated along with the sperm. This fluid contains
hormones (including both testosterone and estrogens), enzymes, and special nutrients that are
essential for sperm maturation (Guyton and Hall, 2010; Ganong, 2011; Fox, 2013).
The normal motile, fertile sperm are capable of flagellated movement through the fluid
medium. The activity of sperm being greatly enhanced in a neutral and slightly alkaline
medium, but it is greatly depressed in a mildly acidic medium. A strong acidic medium can
cause rapid death of sperm. The activity of sperm increases markedly with increasing
temperature, but so does the rate of metabolism, causing the life of the sperm to be considerably
shortened (Ganong, 2011).
2.1.3 The seminal vesicle
Each seminal vesicle is a tortuous, loculated tube, lined with a secretory epithelium that secretes
a mucoid fluid containing an abundance of fructose, citric acid, and other nutrient substances,
as well as large quantities of prostaglandins and fibrinogen (Costanzo, 2006). During the
process of emission and ejaculation, each seminal vesicle empties its contents into the
ejaculatory duct shortly after the vas deferens empties the sperm (Fox, 2013). This adds greatly
to the volume of the ejaculated semen, and the fructose and other substances in the seminal
fluid are of considerable nutrient value for the ejaculated sperm until one of the sperm fertilizes
the ovum (Costanzo, 2006). According to Guyton and Hall (2010) and Costanzo (2006),
prostaglandins are believed to aid fertilization in the following two ways:
(1) by reacting with the female cervical mucus to make it more receptive to sperm movement
and
(2) by possibly causing backward, reverse peristaltic contractions in the uterus and Fallopian
tubes to move or direct the ejaculated sperm toward the ovaries.
9 2.1.4 The prostate gland
The prostate gland secretes a thin, milky fluid that contains calcium, citrate ion, phosphate ion,
a clotting enzyme, and a profibrinolysin (Costanzo, 2006; Guyton and Hall, 2010). During
emission, the capsule of the prostate gland contracts simultaneously with the contractions of
the vas deferens so that the thin, milky fluid of the prostate gland adds further to the bulk of
the semen (Costanzo, 2006). The slightly alkaline characteristic of the prostatic fluid may be
quite important for successful fertilization of the ovum, as the fluid of the vas deferens is
relatively acidic, owing to the presence of citric acid and metabolic end products of the sperm
and, subsequently, helps to inhibit sperm fertility (Ganong, 2011). The vaginal secretions of
the female are also acidic. Sperm do not become optimally motile until the pH of the
surrounding fluids rises to between 6.0 and 6.5. Consequently, it is probable that the slightly
alkaline prostatic fluid helps to neutralize the acidity of the other seminal fluids during
ejaculation, and thus enhances the motility and fertility of the sperm (Ganong, 2011).
2.1.5 Semen
Semen, which is ejaculated during mating, is composed of the seminal fluid and sperm cells.
Seminal fluid arises from the sex glands, such as the seminal vesicles, the prostate gland, and
the bulbourethral glands (Ganong, 2005; Costanzo, 2006; Berne and Levy, 2008). The bulk of
the semen fluid is from the seminal vesicles, which is the last to be ejaculated and serves to
wash the sperm through the ejaculatory duct and urethra.
The average pH of the combined semen or seminal fluids is approximately 7.5. The alkaline
prostatic fluid contributing more to neutralize the mild acidity than the other components of
the semen. The prostatic fluid then gives the semen a milky appearance, while the fluid from
the seminal vesicles and mucous glands gives the semen a mucoid consistency (Guyton and
Hall, 2010). A clotting enzyme from the prostatic fluid causes the fibrinogen of the seminal
vesicle fluid to form a weak fibrin coagulum, which retains the semen in the deeper regions of
the vagina, where the cervix is located (Guyton and Hall, 2006). The coagulum dissolves after
a few minutes because of lysis by fibrinolysin formed from the prostatic profibrinolysin. In the
early minutes after ejaculation, the sperm remain relatively immobile, possibly because of the
viscosity of the coagulum. As the coagulum dissolves, the sperm simultaneously become
highly motile (Guyton and Hall, 2010).
10 Although sperm can live for many weeks in the male genital ducts, once they are ejaculated in
the semen, their maximal life span is only 24 to 48 hours at body temperature. At lowered
temperatures, however, semen can be stored for several weeks, and when frozen at
temperatures of -196°C, sperm has been preserved for years (Hafez and Hafez, 2000; Nur et
al., 2010).
2.2 Factors affecting the quality of the sperm
2.2.1 Environment
Photoperiod, temperature, humidity, nutrition, diseases and parasites are some of the
environmental factors that can affect animal production and reproduction (Brito et al., 2002).
An increasing number of reports suggest that chemical and physical agents in the environment,
introduced and spread by for example, human activity, may affect male fertility in humans
(Jerewics et al., 2009). Much has then been done on effects of environmental factors on human
sperm quality and fertility (Duty et al., 2003; Hauser et al., 2003). So for example cattle
reproduction can be affected by heat stress under high ambient temperatures and/or humidity,
the body thermoregulatory mechanisms are unable to increase body heat loss and internal
temperature generally increases above the physiological limits (Brito et al., 2002). In the
tropics, sperm production and semen quality have been shown to decrease during the hot season
(Wolfenson et al., 2000). The elevation of the testicular temperature generally results in an
increased metabolism and oxygen demand, but testicular blood flow is limited and this
increased demand cannot be supplied resulting in hypoxia, the generation of reactive oxygen
species and deterioration of semen quality (Leonardo et al., 2004).The maintenance of the
testicular temperature at 4 to 5 °C below body temperature is essential for normal
spermatogenesis in rams (Leonardo et al., 2004).
2.2.2 Season of the year
Sheep are seasonal breeders (Rosa and Bryand, 2003) and suggestions by Ghalban et al. (2004)
indicate that the optimal male performance may be obtained during the period of increasing
daylight length. In spring and summer both sperm quantity and quality were recorded to be
higher than that in winter or autumn in bucks. Sheep and goats thus exhibit great seasonal
variation in semen quality (Leboeuf et al., 2000). These seasonal variations in both semen
11 quality and quantity are mainly due to changes in daylight length throughout the year (Ghalban
et al., 2004). Optimal reproductive performance of crossbred rams was recorded in late summer
and the beginning autumn by Moghaddam et al, (2012). It was also reported that sperm mass
motility increased steadily from the beginning of the mating season to the end of the mating
season in rams (Ghalban et al., 2004). Photoperiodic signals are generally translated into
stimuli on the reproduction system by changes in the pattern of secretion of melatonin from the
pineal gland (Munyai, 2012). It has been documented by Zamiri et al. (2005), that the highest
values of thyroid stimulating hormone, T4, free T4 index, testosterone, total sperm number,
percentage normal sperm, percentage live sperm, sperm concentration, semen volume and
scrotal circumference were recorded from early summer to winter with the lowest values being
detected at the end of spring.
2.2.3 Age of the male
Increased age of the bull has been associated with decreased sperm motility and increased
minor sperm defects (Brito et al., 2002). Mature rams then generally also have higher ejaculate
volumes, sperm concentrations and total sperm per ejaculate than younger rams (Hafez and
Hafez 2000; Ghalban et al., 2004). According to Salhab et al. (2003), good quality semen can
be collected from growing Awassi rams at 11 months of age. This is in agreement with the
work done by Kumar et al. (2010) who reported that Malpura ram lambs 9-12 months of age
can produce good quality semen.
2.2.4 Nutrition
It has been indicated that adequate nutritional management is crucial for successful
reproduction efficiency in sheep (Smith and Akinbamijo, 2000; Fernandez et al., 2004;
Kheradmand et al., 2006). Carbohydrates, protein and nucleic acid metabolism and their
deficiency may for example impair spermatogenesis and libido in males (Smith and
Akinbamijo, 2000; Alejandro et al., 2002; Mitchell et al., 2003; Kheradmand et al., 2006);
while improved dietary intake of higher energy levels and protein supplementation in rams can
improve the reproductive performance during the breeding season (Kheradmand et al., 2006).
The supplementation of vitamin B12 in semen extenders have been shown to significantly
improved spermatozoa quality, viability, motility, progressive motility, normal spermatozoa
and decreased morphological defects (Hamedani et al., 2013). Vitamin B12 deficiency has been
reported to increase the number of abnormal sperm and decrease the motility and velocity of
12 sperm in male rats (Watanabe et al., 2003). Vitamin B9 (folic acid) may also be vital for proper
development of human sperm, as it is needed for the production of DNA (Wallock et al., 2001).
It has been suggested that in rams, vitamins B1, B6 and B12 play a key role in the
thermoregulation of the scrotal skin and rectal temperature and help maintain libido, semen
quality and fertility during heat stress (Hamedani et al., 2013). Azawi and Hussein (2013) also
reported an increase in the viability of ram spermatozoa diluted in the Tris diluent containing
vitamins C or E (stored at 5˚C, for 120 h).
2.3 The collection of the ram semen
For the collection of semen, which is the first phase, there are certain prerequisites (depending
on the collection technique used) that have to be adhered to. Firstly it is essential to clean and
shave the prepuce of the ram- to prevent any semen contamination during the semen collection
process. Further the area where semen collection takes place must be sheltered and in a dust
free environment. Thus harmful factors such as exposure to sunlight, dust and water (especially
when using the artificial vagina) must be minimized during the semen collection period. Care
should be taken that all equipment to be used are also clean and sterile. Generally there are two
methods that are used for semen collection in rams; these are the use of the artificial vagina
and/or the electrical ejaculator.
2.3.1 The artificial vagina
The artificial vagina (AV) is the most commonly used method of semen collection, as it gives
an ejaculate similar to that obtained during natural mating. The clean, dry, artificial vagina is
assembled and filled with hot water (45 to 50˚C), to create the required temperature and
pressure. A cone with a calibrated semen collection tube is affixed to one end of the AV, while
the lining at the open end is lubricated with some sterile petroleum jelly. An ewe (preferably
in estrus) or a dummy ewe can be used for the semen collection. Generally sexually active
rams display greater levels of investigatory olfactory behavior towards the stimulus females
(Roselli and Stormshark, 2009). The temperament and libido of the ram is very important,
especially during training of the rams to mount and adopt to ejaculate into the artificial vagina.
To obtain good semen sample, the ram must not be allowed to mount the ewe immediately, in
other words teasing the ram before mounting is important. During mounting, the ram’s penis
13 is diverted into the artificial vagina for ejaculation. It is important to keep the semen collection
tube warm (with the hand) during the collection process, so as to prevent cold shock to the
ejaculate.
2.3.2 Electrical stimulation
The electro-ejaculator can be used for rams that are not trained or cannot mount an ewe due to
an injury. In the case of electrical stimulation, the quality of the semen sample also depends
largely on the efficient application of this technique. With electrical stimulation method, semen
density is generally lower per unit volume as compared to the AV since it contains more
seminal fluid or even urine. It was however documented by Hafez and Hafez. (2000) that
electrical stimulation is a crude imitation of the complex natural mechanisms involved with
ejaculate.
The mechanism by which the electro ejaculator operates is that a few electrical impulses (6 to
12V) applied rectally to the ram usually causes the ram to ejaculate. Since the ram exhibits
strong contractions during the application of this technique, it is essential that the ram is firmly
secured, thus labour intensive compared to collection using AV.
The electrode of the ejaculator is firstly lightly smeared with medicinal paraffin to facilitate the
insertion of electrode into the ram’s rectum and improve conductivity. The depth to which the
electrode is inserted; the strength and duration of the stimuli (although varying from one
individual to the next) are essential factors in other to obtain acceptable ejaculate. It is once
again important to keep the semen as close as possible to the body temperature (38˚C), both
during and after semen collection (Hafez and Hafez, 2000).
2.3.3 The principle involved in the use of the artificial vagina and electro-ejaculator
Ejaculation results from inherent reflex mechanisms, integrated in the sacral and lumbar
regions of the spinal cord, and these mechanisms can be initiated by either psychic stimulation
from the brain or actual sexual stimulation from the sex organs, but usually it is a combination
of both (Guyton and Hall, 2010). Ewes in estrus however, sexually transmit stimulating
olfactory cues that together with other sensory and behavioural signals, attract sexually
interested rams (Roselli and Stormshak, 2009). The most important source of sensory nerve
signals for initiating ejaculation is the glans penis. The glans contains a sensitive sensory endorgan that transmits impulses to the central nervous system. The action of mating on the glans
14 stimulates the sensory end-organs, and the sexual signals in turn pass through the pudendal
nerve, through the sacral plexus to the sacral portion of the spinal cord and finally up the spinal
cord to undefined areas in the brain. During natural service, the sensory nerve endings in the
penile integument and the deeper penile tissues are essential for ejaculation (Hafez and Hafez,
2000). Impulses may also enter the spinal cord from areas adjacent to the penis to aid in the
stimulation of ejaculation (Guyton and Hall, 2010). So for instance, stimulation of the rectal
epithelium, the scrotum, and perineal structures in general may send signals into the spinal cord
that add to the sexual sensation. Sexual sensations can even originate in internal structures,
such as in areas of the urethra, bladder, prostate, seminal vesicles, testes, and vas deferens.
Indeed, one of the causes of libido is filling of the sexual organs with secretions. The concern
is that the rams should not be stressed, but rather be stimulated to provide semen that is similar
to that obtained from natural mating.
Penile erection is the first sign of the male sexual stimulation, and the degree of erection is
proportional to the degree of stimulation, whether psychic or physical (Guyton and Hall, 2010).
This sexual stimulation then produces dilation of the arteries supplying the cavernous bodies
of the penis (Fox, 2002). Stiffening and straightening of the penis in ruminants is caused by
the ischiocavernosus muscle which pumps blood from the carvenous space of the crura into the
rest of the corpus cavernosum of the penis (Hafez and Hafez, 2000). Erection is caused by
parasympathetic impulses that pass from the sacral portion of the spinal cord through the pelvic
nerves to the penis. During sexual stimulation, the parasympathetic impulses, in addition to
promoting erection, cause the urethral glands and the bulbourethral glands to secrete mucus.
This mucus then flows through the urethra during mating to aid in the lubrication. Most of the
lubrication is provided by the female sexual organs, rather than by the male. Without
satisfactory lubrication, the male ejaculation is seldom successful due to friction resulting in
painful sensations that inhibit rather than excite sexual sensations (Fox, 2002).
According to Guyton and Hall (2011), when the sexual stimulus becomes extremely intense,
the reflex centers of the spinal cord begin to emit sympathetic nerve impulses that leave the
cord at the T-12 to L-2 vertebrae and pass to the genital organs through the hypogastric and
pelvic sympathetic nerve plexuses to initiate emission, the forerunner of ejaculation. Emission
begins with contraction of the vas deferens and the ampulla to cause expulsion of sperm into
the internal urethra. Then contractions of the muscular coat of the prostate gland are followed
by contraction of the seminal vesicles, expelling the prostatic and seminal fluid into the urethra
15 and forcing the sperm forward. All these fluids mix in the internal urethra with mucus already
secreted by the bulbourethral glands to eventually form semen.
The filling of the internal urethra with semen elicits sensory signals that are transmitted through
the pudendal nerves to the sacral regions of the spinal cord, giving the feeling of sudden fullness
in the internal genital organs. Also, these sensory signals further excite rhythmical contraction
of the internal genital organs and cause contraction of the ischio-cavernosus and bulbocavernosus muscles that compress the bases of the penile erectile tissue. These effects together
cause rhythmical, wavelike increases in pressure in both the erectile tissue of the penis and the
genital ducts and urethra, which then "ejaculate" the semen from the urethra to the exterior. At
the same time, rhythmical contractions of the pelvic muscles and even some of the muscles of
the body trunk cause thrusting movements of the pelvis and penis which also help propel the
semen into the deepest recesses of the vagina and perhaps even slightly into the cervix. As
documented by Hafez and Hafez (2000), ejaculation is the passage of the resultant semen along
the penile urethra.
2.3.4 The hormonal principles
A major share of the control of sexual functions in both the male and the female begins with
the secretion of gonadotropin-releasing hormone (GnRH) by the hypothalamus. This hormone
in turn stimulates the anterior pituitary gland to secrete the gonadotropic hormones: luteinizing
hormone (LH) and follicle-stimulating hormone (FSH). LH being the primary stimulus for
testosterone secretion by the testes, and FSH mainly stimulating spermatogenesis. According
to Roselli and Stormshak (2009), the tonic secretion of testosterone by the testis activates the
copulatory behavior in rams.
The secretion of LH by the anterior pituitary gland is also cyclic, with LH following the
pulsatile release of GnRH. Conversely, FSH secretion increases and decreases only slightly
with each fluctuation of GnRH secretion; instead, it changes more slowly, several hours in
response to longer-term changes in GnRH. Due to the much closer relationship between GnRH
secretion and LH secretion, GnRH is also widely known as LH-releasing hormone.
Testosterone is secreted by the interstitial cells of Leydig in the testes (Guyton and Hall, 2006),
but only when stimulated by LH from the anterior pituitary. Furthermore, the testosterone
quantity secreted increases approximately directly proportion to the amount of LH available.
Testosterone secreted by the testes in response to LH has the reciprocal effect of inhibiting the
16 anterior pituitary secretion of LH. Most of this inhibition probably results from the effect of
testosterone on the hypothalamus to decrease the secretion of GnRH. Thus, testosterone exerts
a homeostatic effect on the hypothalamic-pituitary-gonadal axis through a negative feedback
regulation of GnRH secretion (Scott et al., 2004). Consequently, a decrease in the secretion of
both LH and FSH by the anterior pituitary, and the decrease in LH will reduce testosterone
secretion by the testes. Thus, whenever testosterone secretion becomes too high, this automatic
negative feedback effect reduces testosterone secretion toward the desired operating
circulating level. Conversely, when testosterone is too little, it allows the hypothalamus to
secrete large amounts of GnRH, with a corresponding increase in anterior pituitary LH and
FSH secretion and consequent increase in the testicular secretion of testosterone. FSH binds
with specific FSH receptors attached to the Sertoli cells in the seminiferous tubules. This causes
these cells to grow and secrete various spermatogenic substances. Similarly, the diffusing of
testosterone into the seminiferous tubules from the Leydig cells in the interstitial spaces also
has a strong trophic effect on spermatogenesis.
When the seminiferous tubules fail to produce sperm, secretion of FSH by the anterior pituitary
gland also increases markedly. Conversely, when spermatogenesis proceeds too rapidly,
pituitary secretion of FSH decrease. The cause of this negative feedback effect on the anterior
pituitary is believed to be secretion of inhibin by the Sertoli cells (Guyton and Hall, 2010).
2.4 Evaluation of the semen
Sperm cells are known to be extremely sensitive to air, light, temperature fluctuations, metals,
hypotonic or hypertonic liquids, soap, disinfectants, urine, perspiration and several other
substances. It is therefore imperative to handle the semen as carefully as possible after
collection (Hafez and Hafez, 2000). The microscopic and macroscopic examination of semen
can then be done, based on the following:
2.4.1 Semen volume
Ram semen volume normally varies between 0.5 and 2.0 ml per ejaculate, depending on
collection method used (Hafez and Hafez, 2000).
17 2.4.2 Colour and density of the semen
The normal colour varies from a thick, creamy substance to a clear watery sample, depending
on the density. The colour is thus an indication of the density and also any abnormal colour
indicates contamination of one kind or another. So for example, blood will cause a reddishbrown or pink colour pus cells will show up as grey or brown semen and urine as yellow or
dilute semen (Hafez and Hafez, 2000).
Table 2.1 Relation of semen colour and number of sperm cells (density) per ejaculate in
the ram. (Hafez and Hafez, 2000)
Colour
Number of cells (×109/ml)
Thick creamy (many)
5.0
Creamy
4.0
Thin creamy
3.0
Milky
2.0
Cloudy
0.7
Watery (clear)
insignificant
2.4.3 pH
The normal pH of ram semen varies between 6.4 and 6.7. Variations from these values could
be an indication of an abnormality. So for example when the semen pH is too acidic – it could
be due to too little prostate and seminal fluids or – if the semen is too alkaline – this is an
indication of inflammation (Guyton and Hall, 2011).
2.4.4 Sperm motility and percentage live sperm
With a good semen sample, when the semen collection tube is held against the light, movement
of the semen can be seen with the naked eye. For a more accurate evaluation of sperm motility,
it is however necessary to examine the sample under the microscope (Hafez and Hafez, 2000).
It is essential to examine the semen quality, before the semen is used. It could happen that
sperm cells die because of cold shock or are negatively affected for some or other reason. This
18 makes it essential to examine a drop of semen under the microscope and evaluate the sperm
quality.
Table 2.2 Relationship between sperm motility and the percentage live sperm in the ram
(Zamiri et al., 2010)
5– Very strong progressive, dark waves (90% plus live cells)
4 – Strong progressive undulations (70 to 90% live cells)
3 – Weak undulations (50 to 70% live cells)
2 – Very few, weak, non-progressive undulations (25 to 50% live cells)
1 – No undulations (5 to 25% live cells)
0 – All cells dead
2.4.5 Semen smears
To carry out an examination for the percentage live/dead sperm cells and abnormalities in the
semen, a smear can be made by adding a drop of eosin/nigrosin to a drop of semen on a
microscope slide and examining the smear under the microscope (× 40 magnification). Dead
sperm cells colour red while live sperm cells stay white and abnormalities such as loose heads,
double heads, broken or curved neck and central bodies, as well as curled broken or double
tails, become clearly visible. It is recommended to use only rams with less than 15% abnormal
sperm. To determine the presence of bacteria or pus cells (infection), a drop of sperm can be
stained using giemsa. The pus cells also stain light red (Hafez and Hafez, 2000).
2.5 Dilution of semen
Semen diluents that can be used generally to extend semen samples include pasteurized skim
milk, glucose citrate or tris egg yolk. Dilution of semen is performed slowly, by gradually
adding the diluents drop by drop, mixing it and keeping the mixture close to body temperature.
These diluents serve as buffers, nutritional media and as protectants for the semen. Care must
be taken that the semen does not undergo cold shock during dilution. Cold shock reduces the
membrane permeability to water and solutes, while damaging the acrosomal membrane (Purdy,
19 2006). It was documented by Marco et al. (2005) that only semen with motility score of 4 and
higher can be diluted. By using animal derived additives such as milk or egg yolk in a semen
extender generally implies sanitary risks, not only through the inclusion of specific
microbiological agents, but also by contaminants that may compromise the quality of semen.
An alternative to egg yolk in extenders for ram semen may be soybean lecithin (Gil et al., 2003;
Forouzanfar et al., 2010). Many possible disadvantages of using egg yolk for example bacterial
contamination and variability in sperm survival have been outlined (Aires et al., 2003; Amirat
et al., 2004; Fukui et al., 2008). It was however documented by Munyai (2012) that skim milk
and egg yolk are generally good sources of lipids which generally provide protection of the
sperm membranes to temperature changes. According to Valente et al. (2010), egg yolk as a
supplement is difficult to be replaced in ram semen extenders.
Since 50 to 60 million live sperms are required per insemination, the density of the ejaculate
determines the number of ewes that can be inseminated per ejaculate and also determines the
volume of the insemination dose that must be used. This volume generally varies between 0.05
and 2ml (Hafez and Hafez, 2000).
2.6 Storage of the semen
2.6.1 Liquid semen
A wide variety of diluents can be used for storing liquid semen, for example tris-fructose egg
yolk, pasteurized skim milk-egg yolk, glucose citrate-egg yolk and sodium citrate. As
documented by Forouzanfar et al. (2010), an extender should contain an energy source (glucose
or fructose). Agents that comprise good extending media should be able to provide nutrients as
source of energy, protect against harmful effect of rapid cooling, provide a buffer to prevent
harmful shifts in pH as lactic acid is formed, maintain the proper osmotic pressure and
electrolyte balance, inhibit bacterial growth and protect sperm cells during freezing (Hafez and
Hafez, 2000). Antibiotics are also frequently added to the semen diluent for the purpose of
destroying any possible micro-organisms, while glycerol serves as a cryoprotective agent in
the semen freezing process (Munyai, 2012). The cryoprotectant prevents the crystallization of
water within the sperm cells, which ultimately allows the sperm cells to be frozen rapidly (Holt,
2000; Munyai, 2012).
The best results have been obtained when storing liquid semen by
adding 500 to 1000 µg streptomycin to 1ml of diluted semen. Marco (2005) reported that
20 diluted semen can be stored successfully for up to 3 days at a temperature of 2 to 5˚C, or for
24 hrs at 18 to 20˚C.
However this method of liquid semen preservation is not very desirable as fertility is generally
lower than with fresh, diluted semen. It is still a sound practice to collect semen when it is
required (Hafez and Hafez, 2000).
2.6.2 Frozen semen
Cryopreservation as a technique for the long term storage of semen has many advantages, but
the freezing and thawing processes generally induce detrimental effects in terms of ultrastructural, biochemical and functional sperm damage (Watson, 2000; Munyai, 2012). Resulting
in a decrease of sperm motility, membrane integrity and fertilizing ability (Purdy, 2006;
Munyai, 2012) Semen cryopreservation induces the formation of intracellular ice crystals,
osmotic and chilling injury that gives rise to sperm damage like cytoplasmic fractures, an effect
on the cytoskeleton and genome related structures (Isachenko, 2003). The membrane
permeability is increased after cooling, and may be a consequence of increased membrane
leakiness and specific protein channels. Cooling also affects calcium regulation and this has
severe consequences on the cellular function, including cell death (Munyai, 2012). When
proper cooling procedures are used, these consequences can be avoided.
The use of
programmable freezer have been reported with better sperm motility (Hammadeh et al., 2001;
Clulow et al., 2008) and with less cryo-damage to the sperm cell (Petyim and Choavaratana,
2006).
There are generally two ways of preserving frozen ram semen, namely pellets or straws. In
pellet preservation, the diluents tris-fructose-egg yolk- glycerol or raffi-nose-egg yolk-glycerol
or lactose-egg yolk-glycerol are used. Egg yolk being the main component in the extenders for
storage and cryopreservation of semen in most mammalian species, including bull, ram goat,
pig and even human semen (Forouzanfar et al., 2010). Egg yolk has been shown to have
beneficial effects on sperm cryopreservation survival as a protector of the sperm plasma
membrane and acrosome against temperature related injury, in association with other
components because of the lipids that it contains (Purdy, 2006). Glycerol plays a vital role as
a protectant during freezing. Gil et al. (2003) reported that glycerol, despite its value as a
cryoprotectant, is metabolically toxic to spermatozoa and noxious to membrane integrity
depending on the concentration and the temperature at which it is added. Thus calling for a
compromise if optimal results are to be achieved. The toxicity of glycerol thus limits the use
21 of high concentrations of glycerol in the cryopreservation media (Forouzanfar et al., 2010).
Sodium citrate, milk based semen extenders (non-fatty milk powder and distilled water) and
tris-based extenders, including egg yolk were for example used by Paulenz et al. (2002) and
sperm viability parameters were shown to be influenced by storage time and the extender.
Valente et al. (2010) noted that the semen extender composition has a major effect on postthawed sperm viability. Tris-egg yolk based diluents have been reported to provide adequate
cryoprotection (Salamon and Maxwell, 2000; Valente et al., 2010).
In a frozen form, semen can then be stored in liquid nitrogen (- 196˚C) indefinitely. When
required, it can be thawed at a temperature of 38 or 60 or 70˚C depending on the freezing
method, for a period of approximately 45 seconds, where after, it is placed in the water bath at
32˚C. The dose used for AI using frozen ram semen varies between 0.1 and 0.2 ml with a
density of 150 to 180 million live sperms per insemination (Hafez and Hafez, 2000).
When inseminating using frozen semen, it is desirable to keep the sperm numbers that are being
inseminated as high as possible, since sperm viability decreases rapidly after thawing. At
present, the conception rate achieved with frozen semen (following cervical insemination) in
sheep is low and use is made of the technique of intra-uterine insemination with the aid of the
laparoscopy. In this technique, semen is directly deposited in the uterine horns to significantly
enhance the chances of conception, even though less sperm are inseminated with this technique.
Suitable semen extenders to preserve an adequate number of spermatozoa with all the attributes
needed to overcome the cervical barrier and to improve fertilization post thawing are needed
to achieve these goals (Gil et al., 2003). This is enunciated by the recent work in that a slight
decrease of post thaw ram sperm abnormalities are achieved with the use of high levels of
sugars and extenders containing trehalose and raffinose (Jafaroghli et al., 2011). So it has been
confirmed that it is possible for ram semen to be successfully cryopreserved in straws (Sabev
et al., 2006; García-Álvarez et al., 2009b) and so for example lot of research has been done on
ram semen preservation with good results (Gil et al. 2000; Holt, 2000; Salamon and Maxwell.
2000; Gil et al., 2003; Marco-Jimémenez et al., 2005; Pereira et al., 2009; Nur et al., 2010;
Valente et al., 2010; Moustacas et al., 2011).
22 2.6.2.1 Semen freezing procedures
Resent work seems to have incorporated different number of ram semen freezing protocols.
The examples of protocols were as follows:
Protocol 5 ˚C (P5 ˚C) (Gil et al., 2003)
1. Cooling to 5˚C within 60 min in the waterbath.
2. Extend semen at 5˚C with f/2 of each extender to 0.8 × 109cells/ml. (Where f/2 refers
to two fractions of glycerol)
3. Equilibration at 5˚C for 2 hr.
4. Packaging in 0.25ml mini straws at 5˚C.
5. Freezing.
Protocol 15 ˚C (P15 ˚C) (Gil et al., 2003)
1. Cooling to 15 ˚C within 30 min.
2. Extend semen at 15 ˚C with f/2 of each extender to 0.8 × 109 cells/ml. (Where f/2 refers
to two fractions of glycerol)
3. Further cooling to 5 ˚C within 30 min in a waterbath.
4. Equilibration at 5 ˚C for 1.5 hr.
5. Packaging in 0.25ml mini-straws at 5 ˚C.
6. Freezing.
According to Gil et al. (2000) the freezing protocols were as follows:
Protocol 1:
Centrifugation before filling the straws to re-concentrate the diluted semen to a calculated
sperm concentration of 800 × 106 sperm/ml.
1. Further dilution (22 ˚C) to a final ratio of 1+4 semen/diluents using fraction 1 of the
extender (without the cryoprotectant).
2. Cooling to 5˚C within 1 hr.
3. A second dilution at 5˚C to double the volume with fraction 2 of the extender (extender
with the cryoprotectant)
4. Equilibration at 5˚C for 2 hr.
5. Centrifugation at 5˚C (700g/10 min).
23 6. Removing supernatant to yield a calculated final sperm concentration of 0.8 × 109
sperm/ml.
7. Packaging in 0.25ml mini-straws at 5˚C.
8. Freezing.
Protocol 2:
Involved an appropriate ejaculate extension to yield 800 × 106 sperm/ml
1. Further dilution (22 ˚C) to 1.6 × 109 cells/ml with diluents fraction 1.
2. Cooling to 5˚C within 1 hr.
3. A second dilution at 5˚C to 0.8 × 109 sperm/ml with diluents fraction 2.
4. Cooling to 5˚C within 1 hr.
5. Packaging in 0.25ml mini-straws at 5 ˚C.
6. Freezing.
2.6.3 Cryoprotective agents
Cryoprotectants are used in the cryopreservation medium to reduce the physical and chemical
stresses derived from cooling, freezing and thawing on the sperm cells (Purdy, 2006).
Cryoprotectants can be classified as penetrating or non-penetrating agents, where the
penetrating agents or intracellular cryoprotectants including glycerol, dimethyl sulphoxide,
ethylene glycol and propylene glycol have low molecular weights and induce membrane lipid
and protein re-arrangement, resulting in increased membrane fluidity, greater dehydration at
low temperatures, reduced intracellular ice formation and an increased sperm survival rate to
cryopreservation (Holt, 2000). It has been suggested that intracellular ice formation in the
sperm cell is one of the major detrimental factors that reduce the viability and membrane
integrity of frozen thawed sperm (Jafaroghli et al., 2011). Purdy, (2006) reported intracellular
cryoprotectants to be solvents that normally dissolve sugars and salt in the cryopreservation
medium.
Ram semen has been proven to be more difficult to cryopreserve than the semen of other farm
animals (Nur et al., 2010). Despite advances in the cryopreservation of mammalian
spermatozoa, there has been less success in ram spermatozoa cryopreservation than with bull
spermatozoa (Varisli et al., 2008). The basis of the cryoprotective properties of glycerol is not
completely understood (Aires et al., 2003). It has been documented by Nur et al. (2010) that
the presence of glycerol lowers the quality and fertilizing capacity of semen. However, as
24 mentioned earlier, the use of cryoprotactants is obligatory for maintaining the post-thaw
cryosurvival of ram semen.
2.7 In vitro fertilization
In the natural situations, the female reproductive tract is an excellent environment, not only for
fertilization, but also for development and maturation of the fertilized ovum. For these to
happen, a number of reproductive physiological activities are of vehement importance.
Reproduction begins with the development of the ova in the ovaries. In the middle of each
sexual cycle, a single ovum is expelled from an ovarian follicle (ovulation) into the abdominal
cavity near the open fimbriated ends of the two fallopian tubes of an ewe. This ovum then
passes through one of the fallopian tubes into the uterus; if it has been fertilized by a sperm cell
in the isthmic junction of infundibulum, it gets implanted in the uterus (Fox, 2002), where
further development into a fetus will take place (Ganong, 2005; Costanzo, 2006).
During the fetal life, the outer surface of the ovary is covered by a germinal epithelium, which
embryologically is derived from the epithelium of the germinal ridges. As the female fetus
develops, primordial ova differentiate from this germinal epithelium and migrate into the
substance of the ovarian cortex (Guyton and Hall, 2010). Each ovum then congregates around
it a layer of spindle cells from the ovarian stroma (the supporting tissue of the ovary) and causes
them to take on epithelioid characteristics, these cells are then called granulosa cells. The ovum
surrounded by a single layer of granulosa cells is called a primordial follicle. The ovum itself
at this stage is still immature, requiring two more cell divisions before it can be fertilized. At
this time, the ovum is called a primary oocyte (Fox, 2013). During all the reproductive years
of adult life, the primordial follicles develop enough to expel their ovum each sexual cycle; the
remainder degenerate or become atretic. At the end of reproductive capability, only a few
primordial follicles remain in the ovaries, and even these degenerate soon thereafter. All these
activities are regulated hormonally (Fox, 2002).
Gonadotrophic hormones are secreted from anterior pituitary gland (Costanzo, 2006; Fox,
2013). Secretion of most of these anterior pituitary hormones is controlled by gonadotropin
releasing hormones (GnRH) formed in the hypothalamus and then transported to the anterior
pituitary gland by way of the hypothalamic-hypophysial portal system (Ganong, 2005; Guyton
25 and Hall, 2010). The gonadotrophic hormones are luteinizing hormone (LH) and follicular
stimulating hormone (FSH).
Estrogen which is secreted by the follicles in small amounts has a strong effect to inhibit the
production of both LH and FSH (Fox, 2013). Even when there is an availability of progesterone
(secreted by ovaries), the inhibitory effect of estrogen is multiplied, even though by itself
progesterone has little effect. These feedback effects seem to operate mainly on the anterior
pituitary gland directly, but also operate to a lesser extent on the hypothalamus to decrease the
secretion of GnRH, especially by altering the frequency of the GnRH pulses (Fox, 2002;
Guyton and Hall, 2010; Fox, 2013).
In addition to the feedback effects of progesterone and estrogen, other hormones are also
involved. So for example inhibin is secreted, along with the steroid sex hormones, by the
granulosa cells of the ovarian graafian follicle in the same way that the Sertoli cells secrete
inhibin in the male testes (Fox, 2006). Inhibin hormone has the same effect in the female as in
the male, inhibiting the secretion of FSH and, to a lesser extent LH by the anterior pituitary
gland (Fox, 2002; Fox, 2006; Guyton and Hall, 2010; Fox, 2013). Therefore, it is believed that
inhibin may be especially important in causing the decrease in secretion of FSH and LH at the
end of the female sexual cycle.
2.8 The fertilization process
Fertilization of the ovum normally takes place in the isthmic junction of the infundibulum of
one of the fallopian tubes, soon after both the sperm and the ovum reach the site (Ganong,
2005; Costanzo, 2006; Guyton and Hall, 2010). However before a sperm can penetrate the
ovum, it must first penetrate the multiple layers of granulosa cells attached to the outside of the
ovum (the corona radiate) and then bind to and penetrate the zona pellucida surrounding the
ovum proper (Fox, 2002; Ganong, 2005; Fox, 2006; Berne and Levy, 2008; Guyton and Hall,
2010; Fox, 2013).
Once a sperm has entered the ovum, the oocyte divides again to form the mature ovum plus a
second polar body that is expelled. The mature ovum still carries 23 chromosomes in its
nucleus.
26 In the meantime, the morphology of fertilizing sperm has also changed. On entering the ovum,
the head swells to form a male pro-nucleus. Later, the 23 unpaired chromosomes of the male
pro-nucleus and the 23 unpaired chromosomes of the female pro-nucleus align themselves to
re-form a complete complement of 46 chromosomes (23 pairs) in the fertilized ovum (Guyton
and Hall, 2010).
2.9 Sperm capacitation
There are certain natural changes that spermatozoa must undergo (capacitation) so as to fertilize
the ovum. Although spermatozoa are said to be mature when they leave the epididymis, their
activity is held in check by multiple inhibitory factors secreted by the genital duct epithelia.
Therefore, when sperm are first expelled in the semen, they are unable to penetrate the ovum.
However, on coming in contact with the fluids of the female genital tract, multiple changes
occur that activate the sperm for the final processes of fertilization. These collective changes
are called capacitation of the spermatozoa. According to Guyton and Hall (2010) some changes
that are believed to occur are as follows
1. The uterine and fallopian tube fluids wash away the various inhibitory factors that
suppress sperm activity in the male genital ducts.
2. While the spermatozoa remain in the fluid of the male genital ducts, they are continually
exposed to many floating vesicles from the seminiferous tubules containing large
amounts of cholesterol. This cholesterol is continually added to the cellular membrane
covering the sperm acrosome, toughening this membrane and preventing release of its
enzymes. After ejaculation, the sperm deposited in the vagina swim away from the
cholesterol vesicles upward into the uterine cavity, and gradually lose much of their
other excess cholesterol over the next few hours. In so doing, the membrane at the head
of the sperm (the acrosome) becomes much weaker.
3. The membrane of the sperm also becomes much more permeable to calcium ions, so
that calcium now enters the sperm in abundance and changes the activity of the
flagellum, giving it a powerful whiplash motion in contrast to its previously weak
undulating motion. In addition, the calcium ions cause changes in the cellular
membrane that covers the leading edge of the acrosome, making it possible for the
27 acrosome to release its enzymes rapidly and easily as the sperm penetrates the granulosa
cell mass surrounding the ovum and even more so as it attempts to penetrate the zona
pellucida of the ovum itself. Thus, multiple changes occur during the process of
capacitation. Without these, the sperm cannot make its way to the interior of the ovum
and induce fertilization.
Stored in the acrosome of the sperm are large quantities of hyaluronidase and proteolytic
enzymes. Hyaluronidase depolymerizes the hyaluronic acid polymers in the intercellular
cement that hold the ovarian granulosa cells together (Fox, 2002; Fox, 2006; Guyton and Hall,
2010; Fox, 2013). The proteolytic enzymes digest the proteins in the structural elements of
tissue cells that still adhere to the ovum. When the ovum is expelled from the ovarian follicle
into the fallopian tube, it still carries with it multiple layers of granulosa cells. However, before
a sperm can fertilize the ovum, it must dissolve these granulosa cell layers, and then penetrate
through the thick covering of the ovum itself, the zona pellucida (Fox, 2002; Fox, 2006; Berne
and Levy, 2008; Guyton and Hall, 2010; Fox, 2013). To achieve this, the stored enzymes in
the acrosome begin to be released. It is believed that of these enzymes hyaluronidase is the
most important in opening pathways between the granulosa cells so that the sperm can reach
the ovum.
When the sperm reaches the zona pellucida of the ovum, the anterior membrane of the sperm
itself binds specifically with receptor proteins in the zona pellucida (Ganong, 2005; Berne and
Levy, 2008; Guyton and Hall, 2010). Then, rapidly the entire acrosome dissolves, and all the
acrosomal enzymes are released (Ganong, 2005). Within minutes, these enzymes open a
penetrating pathway for passage of the sperm head through the zona pellucida to the inside of
the ovum. Within another few minutes, the cell membranes of the sperm head and of the oocyte
fuse with each other to form a single cell (Fox, 2002; Fox, 2006; Guyton and Hall, 2010; Fox,
2013). At the same time, the genetic material of the sperm and the oocyte combine to form a
completely new cell genome, containing equal numbers of chromosomes and genes from ewe
and ram as discussed earlier. Thereafter the embryo begins to divide and develop.
The analysis of semen parameters cannot confirm the quality of semen with certainty, as one
cannot be sure whether the sperm will fertilize or not (Mocé and Graham, 2008). Some semen
evaluation techniques such as sperm binding, oocyte penetration, and in vitro fertilization can
estimate functional aspects of the spermatozoa (Flowers, 2009). Following the work of Hafez
and Hafez. (2000), classic semen evaluation techniques, as well as new automated semen
28 assessment technologies have yielded only estimates of the potential fertilizing capacity of
spermatozoa. In addition, Petrunkina et al. (2007) proposed several criteria for the assessment
of functional sperm parameters regarding capacitating ability. It has been suggested that the
type of semen extender affects the in vitro fertilization potential, while it has no effect on the
developmental potential up to the blastocyst stage (Forouzanfar et al., 2010). To date, reports
arising from research has revealed the role of sperm cryopreservation for both in vivo and in
vitro production of human and animal embryos (Byme et al., 2000; Yildiz et al., 2007;
Forouzanfar et al., 2010). So for example, in sheep lot of research on sperm cryopreservation
for in vitro embryo production has been done successfully (García-Álvarez et al., 2009a;
García-Álvarez et al., 2009b; Periera et al., 2009; Valente et al., 2009; Forouzanfar et al., 2010;
Valente et al., 2010).
For in vitro fertilization to take place, a number of processes are involved. Oocytes have to be
collected washed and matured. Oocyte collection is usually performed by the isolation of
follicles, dissection, aspiration or slicing of follicles or oviductal flushing (Gordon, 2003).
Aspiration as such entails that all visible ovarian follicles are aspirated using a hypodermic
needle, attached to a disposable syringe. The slicing technique involves the use of a surgical
blade, making an incision over the entire ovarian surface into a Petri dish containing a
harvesting medium (Wani et al., 2000; Wang et al., 2007). Dissection again is the method of
dissecting the intact antral follicles with a subsequent carefully controlled rapture to recover
morphologically acceptable oocytes with the least disruption to the surrounding cumulus cells.
Laparoscopic ovum pick-up is another method of recovering the oocytes but from ovaries of
live animals by aspiration through the use of laparoscope (Gordon, 2003).
The follicular oocytes are commonly recovered from ovaries of slaughtered animals (Wani,
2002) and are thereby heterogeneous in quality and developmental competence (RodriguezGonzalez et al., 2002; Gordon, 2003; Bhojwani et al., 2007). Compact cumulus oocyte
complexes (COC’s) are then often recovered from slaughterhouse collected bovine ovaries
(Bhojwani et al., 2007). Oocytes characterized by a decreased developmental competence have
a low ability to reach the embryonic stage and their development is generally disturbed
(Nemcova et al., 2006; Petro et al., 2012).
According to Thibier (2004), with in vitro embryo production, one of the main biohazards
concerning embryos is the repeatability in the donor female and the collection method of
oocytes used, followed by maturation, fertilization and embryo development. The method of
29 COC recovery may influence the degree of heterogeneity of the recovered oocytes (Bhojwani
et al., 2007) and can also affect the efficiency of IVEP (Katska-Ksiazkiewicz et al., 2007).
From the slicing method of oocyte collection from ovaries, COCs may be recovered not only
from the antral follicles on the surface, but also from smaller antral follicles from the inside of
the ovary, which may be in the earlier stages of follicular development after antrum formation
(Bhojwani et al., 2007) while the follicle aspiration method is a preventive sanitary measure in
the in vitro embryo production process (Marius et al., 2009). However, slicing is the most
useful technique of oocyte collection, even though it does not allow for selection according to
follicular diameter (Rodriguez-Gonzalez et al., 2002). Following the work of Yang et al.
(2012), a person should use caution when relying on a particular size of follicle to predict
oocyte maturity. Slicing or aspiration has then generally been used for oocytes recovery in
slaughtered sheep (Wani et al., 2000).
Higher quality oocytes percentages have been reported (80.5 ± 2.4% and 79.8 ± 1.9%) when
using the aspiration method as opposed to lower quality oocytes percentages (42.7 ± 2.3% and
41.2 ± 4.8%) for the slicing method (Marius et al., 2009). It should however be noted that
several researchers from different countries such as Dublin, Denmark, Itally, Russia, USA,
India and Spain have documented high yield of oocytes when using the slicing technique
(Gordon, 2003).
Reports regarding ovum pick-up (OPU) has suggested that the technique is of high value more
in India, where cow slaughter is banned for religious reasons, and there is no opportunity for
researchers to obtain oocytes from the abattoir bovine ovaries, as in most other countries
(Gordon, 2003). It was demonstrated by Manik et al. (2002), that the use of OPU is useful in
recovering developmentally competent oocytes. In cattle, a great number and better quality
oocytes were recovered from Gir cows (Sales et al., 2010) and a high blastocyst rate obtained
(Snjiders et al., 2000; Blondin et al., 2002; Rizos et al., 2005). Lonergan (2010) suggested that
one way of experimentally separating potential issues surrounding the follicle and/or oocytes
for issues relating to the reproductive tract environment, is to use transvaginal ovum pick-up,
coupled with IVF. However, it has been reported that certain factors such as the energy
deficiency and lactation period have negative effects on oocyte quality and endocrine levels,
when using OPU (Gwazdauskas et al., 2000; Walters et al., 2002; Leroy et al., 2005).
Studies done in women demonstrated a higher live birth rate when mature oocytes were
obtained at the time of the retrieval for the natural cycle of IVF/M treatment and also indicated
30 that there may be a higher incidence of miscarriage rate when the transferred embryos were
produced from immature oocytes only (Yang et al., 2012). In addition, in pigs, blastocyst
development was improved by the use of oocytes from sows rather than from pre-pubertal gilts
(Marchal et al., 2001; Sherrer et al., 2004). Embryos collected from pre-pubertal gilts, which
were induced with exogenous hormones, were less likely to form blastocysts in vitro than
embryos collected from natural pre-pubertal gilts (Peters et al., 2001). Sow oocytes also
recorded a higher maturation rate and a lower sperm penetration rate than gilt oocytes (Marchal
et al., 2001).
Studies of bovine and ovine in vitro produced embryos indicated limited success when prepubertal females provide oocytes (Sherrer et al., 2004). However the staining of cumulusoocytes complexes using brilliant cresyl blue (BCB) stain before in vitro maturation could be
used to select developmentally competent oocytes for in vitro embryo production and even for
nuclear transfer (Rodriguez-Gonzalez et al., 2002; Bhojwani et al., 2007; Piotrowaska et al.,
2013).
2.10 Factors affecting oocyte quality
A matter of great importance in attempting to produce animal embryos in the laboratory is the
ability of oocytes to undergo fertilization and develop into an embryo and this ability must
extent all the way through to the transfer and birth of a live animal. Determination of oocyte
quality is generally performed by assessing the ability of oocytes to mature, to be fertilized and
give rise to normal offspring (Duranthon & Renard, 2001; Hussein et al., 2006; Sirard et al.,
2006). The utilization of the recovered oocytes must be for the production of viable embryos,
without reducing their developmental competence (Wani et al., 2000; Shirazi et al., 2005;
Morton et al., 2008). There are however several factors that affect oocyte quality such as the
maturation process and the medium used, age of the animal, ovarian follicular size, stage of the
estrous cycle and ovarian morphology, body condition and nutritional status, reproductive
status of the donor and environmental factors (Gordon, 2003).
2.10.1 Maturation process and media used
The maturation of mammalian COC’s is an important step in reaching the full developmental
competence (Grupen and Armstrong, 2010; Jaskowski et al., 2010). The condition of
maturation in vitro must mimic the in vivo environment (Piotrowaska et al., 2013). The
31 concentration and composition of the compounds which supplement the culture medium play
a significant role in the achievement of full developmental competence by oocytes (Herrick et
al., 2004; Kim et al., 2005; Song et al., 2010; Piotrowaska et al., 2013).
2.10.2 Age of the donor
The developmental competence of oocytes is determined by age of the donor. To carry out
experiments in sheep, cattle, goats and pig embryo production, greater attention is given to prepubertal animals as the size of their oocytes have a marked influence on their ability to mature
to the metaphase-II phase. A progressive decline in human fertility with advancing age is a
well-known phenomenon (Gordon, 2003). In cattle, research has suggested that the big problem
with oocytes from ovaries of pre-pubertal cows is associated with developmental competence
(Gandolfi et al., 2000; Maclellan et al., 2000; Majerus et al., 2000; Salamone et al., 2000;
Taneja et al., 2000; Salamone et al., 2001; Bhojwani et al., 2007). A similar problem has been
reported in the pig (Kikuchi et al., 2009), buffalo (Yindee et al., 2011), monkeys (Zheng et al.,
2001), as well as in sheep (Catt, 2002; Kochhar et al., 2002).
2.10.3 Ovarian follicular size
Utilizing the growth phase of the first follicular wave for oocyte collection has been reported
to have improved the efficiency of blastocyst production (Machatkova et al., 2000). Moreover,
six complementary DNA clones were identified as being differentially expressed in the
dominant follicles - when suppressive subtractive hybridization was used to investigate the
differential expression of the genes in dominant and subordinate follicles on day 1.3 to 2.3 of
the animal’s oestrous cycle (Sisco et al., 2002).
2.10.4 Body condition and nutritional status
Oocytes derived from the ovaries of animals with a lower body condition score (BCS) are
generally of poor quality. Nutritional status has been reported to be correlated with embryo
survival and is a key factor influencing efficiency in assisted reproductive technologies
(Armstrong et al., 2003; Webb et al., 2004). The under nutrition of donor ewes in an IVEP
programme, resulting in lower body weights and a lower body condition score, has a negative
effect on the oocyte quality, which results in a lower cleavage rate and blastocyst formation
(Borowczyk et al., 2006). Nutrition thus has a significant impact on numerous reproductive
functions, including hormone production, fertilization and early embryonic development
(Boland et al., 2001; Armstrong et al., 2003; Boland and Lonergan, 2005). In sheep, conflicting
32 results have been reported for the effect of low or high energy diets on oocytes quality and
early embryonic development (Boland et al., 2001; Papadopoulos et al., 2001). In cattle,
increased systemic levels of ammonia and urea have been unlikely to disrupt the oviduct
environment to a point where embryo survival would be impaired when the levels of ions and
metabolites in bovine oviductal fluids were quantified (Kenny et al., 2002). Greater
competence has been documented in oocytes of dairy cows with a high body condition score,
compared to those with a low score. A greater rate of follicle development was reported for
cows with a more positive energy balance (Gordon, 2003). The effects of nutrition on oocyte
and embryonic development may thus reflect the general energy balance, but may also be
attributed to the specific nutrients in diets such as vitamins, minerals and other supplements
(Wrenzycki et al., 2000).
2.10.5 Morphology of the ovaries
The intra ovarian environment to which oocytes are exposed can play a major role in
determining their developmental competence. The presence of a dominant follicle in either one
or both ovaries has a negative effect on the competence of ovaries. Ovarian development of
Greenland halibut last for more than one year (Albert et al., 2001; Junquera et al., 2003) as
they spawn annually but successive cohorts of oocytes develop over 2 years (Kennedy et al.,
2011).
2.10.6 Environmental factors and IVEP
It has been reported that the removal of impaired cohorts of follicles during autumn has led to
the earlier emergence of healthy follicles and higher quality oocytes (Roth et al., 2001). The
adverse effect of elevated summer temperatures on oocyte developmental competence was also
previously reported in cattle (AI-Katanani et al., 2002).
2.10.7 Reproductive status of the animal and oocyte competence
The reproductive status of the ewes has been assumed to be a constant variable during the
collection of ovaries at the slaughter house. It was however documented by several studies that
puberty, parturition and lactational status, as well as weaning contribute and have an impact on
the quality of the ovaries and as a result affect the quality of the oocytes. Follicular development
is inhibited during lactation and weaning allows the recruitment and selection of follicles that
will undergo pre ovulatory maturation and ovulation (Quesnel, 2009). It has been further
documented by Quesnel (2009) that lactation inhibits LH secretion by inhibiting gonadotrophin
33 releasing hormone secretion and reducing the pituitary response to gonadotrophin releasing
hormones. However as lactation progress, there is an increase in LH secretion, which allows
the resumption of folliculogenesis. Severe restriction in crude protein or digestible energy has
been shown to delay the post-weaning oestrus, through impairment of gonadotrophin releasing
hormone and LH secretion during lactation (Yang et al., 2000a). Feed or protein restriction
has also been shown to impair the ability of oocytes to be fertilized and develop into an embryo
(Yang et al., 2000b). Thus the developmental stage of the follicles from ovaries of post weaned
ewes would be questionable if reproductive status is not assumed to be a constant variable.
However, suggestions have indicated that seasonal anoestrus involves an increased sensitivity
of the hypothalamus to the oestradiol negative feedback, while high feed intake can modify the
sensitivity of the hypothalamus to oestradiol, considering that ewes are seasonal breeders
(Forcada and Abecia, 2006).
Before puberty, the secretion of sex hormones is very low as documented. Guyton and Hall
(2010) reported that the hypothalamus does not secrete significant quantities of gonadotrophic
releasing hormones. Several studies in humans have indicated problems when the sex
hormones happen to be secreted abnormally (hypogonadism or hypergonadism) by the ovaries
(Martini and Nath, 2009; Guyton and Hall, 2011; Martini et al., 2012). In hypogonadism,
ovarian cycle will not occur normally (Guyton and Hall, 2011) adults cannot produce
functional oocytes (Martini and Nath, 2009; Martini et al., 2012), while in hypergonadism, a
granulosa cell tumor develops in the ovary (Guyton and Hall, 2011).
34 CHAPTER 3
MATERIALS AND METHODS
3.1 Study Area
All trials were conducted at the University of the Free State campus, in Bloemfontein, South
Africa. The study site is located 28.57˚ south longitude, 25.89˚ east latitude and at an altitude
of 1304 m above sea level. The trial was carried out from April (autumn) 2014 to April
(autumn) 2015. The experimental animals were kept at the metabolic building of the university
which is under the management and supervision of the Department of Animal, Wildlife and
Grassland Sciences. The metabolic building was designed specifically for the handling of
research animals (small stock, pigs and poultry). The in vitro fertilization study was conducted
at the Reproduction Physiology Laboratory and the project was approved by the University of
the Free State’ Animal Ethical Committee.
3.2 Experimental animals
Two breeds of rams (Dorper and South African Mutton Merino) were used in the study.
3.2.1 Dorper rams
The Dorper breed originated from the successful crossing of the British Dorset ram and Black
head Persian ewe in 1936 by the Meat Control Board at the Grootfontein College of
Agriculture, Middelburg, South Africa. The Dorper is large, well conformed mutton sheep with
a white or black head and a white body. The breed has an extended breeding season, which is
strictly not seasonally limited. The ewes can produce lambs at almost any time of the year and
attain a lambing rate of 150% per season under optimum grazing conditions. Dorper ewes
produce large quantities of milk and have a good mothering ability. The breed is well adapted
to a variety of climatic grazing conditions, although the breed was originally developed for the
more arid areas of South Africa. However, today the Dorper breed is widespread throughout
all provinces of South Africa and abroad. The Dorper thus performs well under various pasture
and feeding conditions and also reacts most favourably under intensive feeding conditions (De
35 Waal and Combrinck, 2000). The plate below shows Dorper ram which was used as semen
donor.
Plate 3.1 Dorper ram used as a semen donor
3.2.2 South African Mutton Merino rams
This breed was imported to South Africa from Germany in 1932 by the Department of
Agriculture as part of a breeding programme. The breed was originally known as the German
Mutton Merino. Through selection for a better wool quality and body conformation, the
uniqueness of the South African breed was recognized in 1971 when the breed name was
changed to the South African Mutton Merino (SAMM). This Mutton Merino is generally a dual
purpose breed, developed to produce a slaughter lamb at an early age, as well as relatively good
quality wool. The breed is renowned for its fertility, whereby lambing percentages of 150%
and even higher has been obtained with mature ewes weighing 77 kg and rams weighing 127
kg (Buduram, 2004). The Mutton Merino has a renowned mothering ability and rears multiple
births with high weaning weights. The SAMM also have a high milk production, enabling
lambs to maintain a high growth rate and weaning weight for early maturity and hence early
marketing. The breed is generally efficient feed converter and popular in feedlots. It is non36 selective in its grazing habits, and is able to utilize low quality roughage. Its grazing habits
limit energy consumption and better utilization efficiency is maintained allowing for increased
mutton and wool production. The South African Mutton Merino is able to perform under most
climatic conditions (Buduram, 2004). The plate below shows South African Mutton Merino
ram which was used as a semen donor.
Plate 3.2 South African Mutton Merino ram used as a semen donor
3.3 Management of experimental animals
Upon arrival, all eight adult rams recording mean body weight of 69.6 ± 9.2 kg and 92.5 ± 6.1
kg and mean age of 54 ± 4.7 months and 36 ± 2.1 months (Dorper and SAMM respectively)
were allowed two weeks to recover and adapt before any activities were performed. Fresh water
was provided ad libitum and fresh feed in the form of Lucerne was also provided. Vaccination
was done in advance for the prevention of pulpy kidney, pasteurella, tetanus, malignant oedema
or blackquarter. Plate 3.3 below shows how cleaning was done.
37 Plate 3.3 Cleaning process to ensure a healthy environment for the rams
Of these diseases treated for, pulpy kidney (enterotoxamia) is a disease generally caused by the
toxin of Clostridium Perfringes type D (rod shaped), from the intestinal tract due to an
immediate change of nutrition from a poor to a good plain of nutrition. Pasteurellosis as such
is a disease caused by beta-hemolytic, gram-negative, aerobic, non-motile, non-spore forming
coccobacilli in the family Pasteurellaceae, due to a combination of stressors including heat,
overcrowding, exposure to inclement weather, poor ventilation, handling and transportation.
While tetanus is a disease caused by a toxin of bacterium clostridium tetani, due to the gaining
of entrance by bacteria into a wound or damaged tissue. Further malignant oedema is caused
by the toxin Clostridium Septicum from soil and intestinal contents of the animals and normally
occurs through contamination of wounds containing devitalized tissue, soil, or some other
tissue debilitant or through activation of dormant spores. Finally black quarter is a disease
caused by a gram-positive, spore-forming, rod-shaped, anaerobic and motile Clostridium
Chauvoei that infect animals through ingestion or wounds associated with lambing, docking,
castration and shearing (Senturk, 2010).
38 The rams were maintained in kraals where each animal was placed in a separate pen. The
cleaning of the kraals was performed early every morning and late in the afternoon. Frequent
hosing of the floors was done daily for proper cleaning and rubber mats were placed in each
pen to provide the necessary bedding and insulation. Two South African mutton merino ewes
were used as teasers for the collection of semen from the rams. The ewes were kept in close
proximity of the rams. The management was routinely similar and under natural ambient
temperature and under natural photoperiod throughout the trial period.
The rams were fed a maintenance diet daily to avoid animals becoming too obese or losing
body weight or condition during the study period. Plate 3.4 below shows water trough that was
placed in each animal pen.
Plate 3.4 Water trough placed in each pen
During the first two weeks of the adaptation period, the experimental rams were fed a
maintenance pellet diet, mixed with ground Lucerne with the purpose of reducing the stress
and facilitating the adaptation process, as well as keeping the rumen functional (Malejane,
2013).
After the adaptation period, the animals were regularly given 2.5 kg of pellets per day (half
was given between 7:00 and 8:00 and the other half between 14:30 and 15:30). Visual
examinations of all animals were done daily for any clinical sign that could indicate illness.
39 3.4 Preparation of the semen extenders
All the chemicals were supplied by Sigma – Aldrich, Co (St. Louis, MO, USA) unless
otherwise indicated. Distilled water was obtained from the Animal Nutrition Laboratory at the
University of the Free State and all the semen extenders were prepared a day before semen
collection. The two semen extenders used were a Tris-egg yolk based extender and a Sodium
Citrate-egg yolk based extender. Different fractions were prepared for each extender, the one
without glycerol (diluent A) and the other containing glycerol (diluent B), for the two step
dilution procedure during semen processing (Gil et al., 2003). After preparation as shown in
plate 3.5 below, the semen extenders were stored at 5˚C, until utilized.
Plate 3.5 Preparation of extenders for semen cryopreservation
3.5 Semen collection and quality evaluation
Prior to semen collection, a teaser ewe was restrained (Plate 3.6) to minimize the movement
and facilitate semen collection.
40 Plate 3.6 Restraining a teaser ewe inside the semen collection pen
The artificial vagina (AV) was then prepared, which briefly entails filling the artificial vagina
with water between 50˚ C and 60˚ C (Plate 3.7). The pressure in the AV was adjusted with the
aid of the amount of water used. The one end of AV was lubricated with a sterile petroleum
jelly (excess lubrication of the AV being avoided as this could result in semen contamination).
The temperature inside the AV was checked prior to collection using thermometer and this
temperature generally ranged between 42˚C and 45˚C. Thereafter the pre-warmed (32˚C)
semen collection tube was fitted to the other end of the AV and kept covered to maintain the
temperature of the collection tube (Hafez and Hafez, 2000).
41 Plate 3.7 Preparation of the artificial vagina before semen collection
Now the ram was then allowed into the collection pen, and after mounting the teaser ewe, its
penis was diverted into the AV with the necessary care not to cause any injury or contamination
(Plate 3.8). When the ram’s penis made contact with the warm, lubricated surface of the AV,
the ram generally gave a forward thrust and ejaculated. A vigorous upward and forward thrust
generally signified ejaculation. Only semen collected in the collection tube was evaluated, as
semen in the surface of AV lining generally resulted in thermal damage to the sperm cells
(Mitchell and Doak, 2004). This ejaculation generally occurred within 1 to 2 seconds. Semen
was deposited into the collection tube and the tube then removed from the AV and semen was
ready for evaluation.
42 Plate 3.8 Collection of semen from the rams using the artificial vagina
3.5.1 Semen evaluation
3.5.1.1 Colour of the ejaculate
The first parameter of the semen that was recorded is colour as this is an indication of the
concentration of an ejaculate and also a means of checking for any contamination of the semen.
3.5.1.2 Semen volume
The volume of the ejaculate was recorded directly from the calibrated collection tube,
immediately after collection. Care was taken to make sure that the tube was kept warm and in
an upright position during readings.
3.5.1.3 Semen wave motion
To assess the sperm wave motion, 10 µl of semen was drawn with a pipette and placed on a
pre-warmed microscope slide (32˚C) and observed microscopically under low magnification
(Olympus) (x 10 magnification). The wave motion was microscopically assessed on the scale
of 0-5. The assessment of the wave motion was done immediately after semen collection (Hafez
and Hafez, 2000).
43 3.5.1.4 Sperm concentration (semen density)
Sperm concentration was determined with the aid of an improved Neubauer haemocytometer
(Plate 3.9) (depth 0.100 mm and 0.0025 mm2). 10 µl of semen was diluted in 990 µl (1: 100)
distilled water, to dilute and also kill the sperm. For the sperm counting, a microscope (x100
magnification) was used. 10 µl of diluted semen was placed on the haemocytometer and
covered with a cover slip. The sample was allowed for the sperm to settle for a period of 5 min.
All visible sperm cells in the 5 designated (5/25) diagonal squires were counted and recorded.
Sperm counting was performed 7 hrs after collection - during that period, the semen mixture
was stored in the refrigerator at a temperature of 5˚C. On the haemocytometer, the 25 large
squares contained 16 smaller squares, which mainly facilitated in the counting of the sperm
cells. The total number of sperm cells per ml was then calculated as the number of sperm cells
counted in the 5 diagonal squares × 5 × 106 sperm/ml (Mitchell and Doak, 2004).
Plate 3.9 Haemocytometer used for sperm counting
3.5.1.5 Sperm viability and morphology
For the evaluation of the percentage live and abnormal sperm, smears were made using the
eosin-nigrosin stain. Briefly 10 µl of semen was diluted with 100 µl of a pre-warmed (32˚C)
eosin-nigrosin stain and a droplet (10 µl) of semen and stain mixture was then placed on a clean
pre-warmed (32˚C) microscope slide and spread evenly with another slide to make a thin semen
44 smear. It is important to push, rather than to pull the edge of the second microscope slide at a
30˚ to 45˚ angle to the first slide excessive pressure is also to be avoided (Malejane, 2013). The
smear was then immediately dried on the warm plate (32˚C) for less than a min. All semen
smear slides were then stored in a clean tray for late determination of the percentage live and
abnormal sperm.
Dead sperm coloured red and abnormalities like loose heads, double heads, broken or bent
necks and mid pieces, double tails, etc, were visible and could be examined microscopically at
×1000 magnification (oil immersion). A representative sample of 100 sperm on each slide from
randomly selected areas was recorded to determine the percentage live (white VS red sperm
cells). The normal VS abnormal sperm were also recorded, specifying the kind of abnormality
of the live sperm.
3.6 Cryopreservation of ram semen
After the collection of semen from two breeds as indicated earlier, one pooled sample of
collected semen was made for each breed (Dorper and South African Mutton Merino) and both
samples divided into two equal halves. Thereafter both samples were diluted with 1:1 v/v, with
fraction A and then again later with 1:1 v/v with fraction B (with glycerol).
The flask (Plate 3.10) was used for the temporary storage of semen samples for the period
during which samples were transported into a cold room, where temperature was decreased
slowly to 5˚C.
45 Plate 3.10 Flask for transportation of semen from collection area to the freezing
Laboratory
After 1 hr, fraction B was added at a dilution rate previously mentioned. After 2 hrs, the diluted
semen samples were loaded into straws (0.25 ml, at 5 °C), sealed with polyvinyl alcohol
powder of two different colours (red and yellow) and transferred to a programmable freezer as
shown in Plate 3.11 (where the temperature was decreased in a stepwise manner). The starting
temperature was 5 °C and semen was cooled to -20 °C at the rate of 1 °C/min. Half of the
semen straws were suspended 3 - 4 cm above liquid nitrogen (Plate 3.12) for 5 min (Jafaroghli
et al., 2011), while the other half was cooled to -80°C at the rate of 5˚C/min in the
programmable freezer. All the semen straws were then plunged directly into liquid nitrogen (196 °C) for storage. Liquid nitrogen tank is shown in Plate 3.13 below.
46 Plate3.11 Semen straws loaded into the programmable freezer for cryopreservation
Plate 3.12 Semen straws suspended 3 – 4 cm above liquid nitrogen vapour for
cryopreservation
47 Plate 3.13 Liquid nitrogen tank used for the storage of semen after cryopreservation
3.7 Thawing of semen for the post-thaw semen analysis
Semen straws were thawed 24 hrs after cryopreservation, by placing the straws into a flask
filled with warm water (37˚C), for 30 seconds (Purdy, 2006; Jafaroghli et al., 2011; Munyai,
2012). Both ends of the semen straws were cut with a pair of scissors and the semen poured
into 10ml pre-warmed test tubes. The microscopic evaluation was performed at four intervals,
starting immediately after thawing, 30 min, 60 min and 120 min post thawing. After every
evaluation, the semen was maintained in an incubator at 37˚C.
48 3.8 In vitro fertilization
3.8.1 Ovary collection
Ovaries were collected at a local abattoir, near the Laboratory and transported to the Laboratory
in sterile saline water (37˚C) in the flask within 3 hrs of slaughter. Immediately on arrival at
the Laboratory, the temperature in the flask was measured and recorded. Ovaries were then
washed three times in a saline (NaCl + Ultrapure water) solution.
3.8.2 Method of oocytes collection
Cumulus oocytes complexes (COC’s) were recovered from the ovarian follicles by aspiration
of the follicular fluid using an 18-gauge needle attached to the 10 ml syringe. The COC’s were
transferred into a 50 ml tube in which the complexes were allowed to settle for 10 minutes,
follicular fluid was poured out and modified phosphate saline (mPBS) was added and allowed
for another 10 minutes to settle, after which the oocytes were transferred to a search dish for
oocyts selection under the microscope. Thereafter, oocytes were washed three times in
modified phosphate saline supplemented with bovine serum albumin (BSA) and three times in
M199, supplemented with fetal bovine serum (FBS). The oocytes were exposed to 26 µM of
brilliant cresyl blue (BCB) diluted in mPBS for 90 minutes at 39˚C in humidified air, with 5%
carbon dioxide. After exposure, the COC’s were transferred into mPBS and washed three times
and then the good quality oocytes according to their cytoplasm colouration (grown oocytes
were those with a blue cytoplasm) were selected under microscope, the selected COC’s were
washed three times in M199 supplemented with FBS, before maturation (Bhojwani et al.,
2007).
3.8.3 Oocytes maturation in vitro
Four-well petri dish containing maturation medium, covered with mineral oil in each well were
pre-incubated for at least 3 hrs before maturation. The maturation medium contained M199
supplemented with FBS and hormones. The COC’s (50 oocytes / well) were then transferred
into a pre-incubated four-well dish and incubated for 24 hrs at 38˚C (5% carbon dioxide and
90% relative humidity). Following maturation, the oocytes were then evaluated under the
microscope and then processed for fertilization.
49 3.8.4 In Vitro Fertilization (IVF)
Three hrs prior to fertilization, Bracket and Oliphant oocytes fertilization medium (BO-IVF)
drops were prepared (5 drops of 100 µl) for washing of oocytes and (7 drops of 50 µl)
fertilization and sperm wash medium were prepared and pre-warmed. At fertilization period,
a frozen semen straw was taken from the liquid nitrogen (LN2) and thawed by dipping the straw
into a flask containing warm water (37˚C), for 30 seconds. The contents of the straw was then
emptied into a 15 ml falcon tube (by cutting both ends of the straw) and the tube filled to 6 ml,
with warm (37˚C) Bracket and Oliphant (BO) sperm washing solution. The frozen-thawed
semen suspension was then centrifuged twice at 1500 rpm for 8 min, at 38˚C. The supernatant
was then carefully aspirated immediately after centrifugation without disturbing the semen
pellet, using a sterile serological pipette. The semen pellet was then diluted with a pre-incubated
BO-Sperm wash solution and a 5 µl aliquot of semen after dilution was placed on a microscopic
slide and covered with a cover slip for the microscopic evaluation of sperm motility - which
was then recorded. At the same time of washing the sperm, the oocytes were also washed in 5
drops of pre-warmed BO-IVF and transferred into 7 drops of BO-IVF for fertilization. Then
50 µl of the prepared semen was placed in the IVF aliquot containing the mature oocytes and
incubated for 18 hrs at 38˚C (5% carbon dioxide and 90% relative humidity) (Bracket and
Oliphant, 1975).
3.8.5 In Vitro Embryo Culture
After 18 hrs of oocyte-sperm incubation, presumptive zygotes were removed from the IVF
aliquots into a 1.5 ml eppendorf tube containing 100 µl of pre-incubated M199 supplemented
with FBS and vortexed for 1.5 min to remove the cumulus cells. After vortexing, zygotes were
washed 3 times in both M199 supplemented with FBS and pre-incubated culture media (SOFBSA) droplets (100 µl) - after which 20 - 25 zygotes were transferred into pre – incubated 50
µl droplet of SOF-BSA medium covered with mineral oil and placed in gas chamber that has
three gases, thereafter the zygotes were incubated at 39˚C for 7 days in a humidified incubator
(5% oxygen, 5% carbon dioxide and 90% nitrogen). During this incubation period, the culture
medium was changed at 48 hrs after fertilization from SOF-BSA to SOF-FBS by aspirating
medium from the aliquots and replacing the medium with the fresh pre-incubated medium with
the aid of a pipette, during which the cleavage rate (2 to 8 cell percentage) was examined and
recorded and embryos were then separated according to number of cell divisions. The media
was changed again on day 5 of incubation. Microscopic evaluations were performed at day 6,
50 and day 7 for the development into the morula and blastocyst stages respectively, and results
were recorded into an excel sheet for data analysis.
3.9 Data Analysis:
The statistical analysis of all collected data for fresh semen (volume, colour, concentration,
motility, viability and morphology); frozen-thawed semen (motility) and performance of the
frozen-thawed semen in vitro (cleavage - 2 to 8 cell) percentages were analyzed using the one
way analysis of variance (ANOVA) procedures of SAS (2009). Tukey’s Studentised Range
(HSD) test (SAS, 2009) was used to determine the differences between treatment means. P
values < 0.05 were considered to be significant.
51 CHAPTER 4
RESULTS
4.1 Effect of breed (Dorper and SAMM) on fresh semen before cryopreservation
The effect of breed (Dorper and SAMM) on fresh ram semen quality before cryopreservation
is set out in Table 4.1. Semen volume and sperm motility recorded significant differences
between the two breeds (P<0.05) with the South African Mutton Merino (SAMM) semen
resulting in higher means than the Dorper semen for the entire observation period of the
experiment. So for example 81.3 ± 0.1% versus 70.2 ± 0.2% and 1.4 ± 0.1ml versus 1.1 ± 0.1ml
for sperm motility and semen volume for the SAMM and Dorper rams were recorded,
respectively. There was no significant difference (P>0.05) recorded between the breeds for
semen colour, concentration, percentage live sperm, percentage dead and the percentage
normal sperm. So for example the general colour of the semen samples for both breeds was
thin creamy.
Table 4.1 Effect of breed on fresh ram semen quality
Breed
Semen parameters (mean±SE)
Motility
Conc.
Live
(%)
(×106/ml)
(%)
Vol.
(ml)
Colour
range
Dorper
1.1±0.1a
3.4±0.4a
70.2±0.2a
2568.3±57.2a
71.2±2.5a
28.8±2.3a 93.7±2.2a
SAMM
1.4±0.1b
3.5±0.3a
81.3±0.1b
2655.8±43.1a
75.8±1.9a
24.5±1.9a 95.2±2.1a
ab
Dead
(%)
Normal
(%)
Values within same column with different superscripts differ significantly (P < 0.05)
4.2 Effect of breed, different extenders and different freezing protocols on the viability of
frozen ram semen for different incubation time periods after thawing
The post-thaw mean sperm motility of the Dorper and SAMM semen frozen using different
extenders and freezing protocols is set out in Table 4.2 and Table 4.3, respectively. The semen
frozen using the liquid nitrogen vapour protocol prior to storage, resulted in a higher sperm
52 motility, compared to semen frozen up to -80˚C (programmable freezer) prior to storage. The
sperm viability of the frozen ram semen was inversely related to a time increase following
thawing. Only the different freezing protocols and time intervals recorded a significant
difference (P<0.05) throughout the entire experiment. Statistically, breeds and the different
extenders recorded no significant difference (P>0.05). However, when the Tukey grouping
was used (studentised range), Tris-egg yolk based extender exhibited a better sperm
performance at the beginning of the experiment than the Sodium Citrate-egg yolk based
extender.
Table 4.2 Mean (±SE) post-thaw sperm motility of Dorper ram semen frozen using
different extenders and freezing protocols at different incubation intervals following
thawing
Breed
Dorper
abcd1/2/3/4
Extender
Freezing
Incubation
protocol (˚C)
after thawing (min)
Tris-based
Vapour (-70)
0
61.4±2.42abc1
Tris-based
-80
0
55.4±2.93abd1
Na Citrate
Vapour (-70)
0
52.2±2.350abc1
Na Citrate
-80
0
50.8±3.51abd1
Tris-based
Vapour (-70)
30
52.0±3.00abc2
Tris-based
-80
30
48.2±3.12abd2
Na Citrate
Vapour (-70)
30
48.0±3.00abc2
Na Citrate
-80
30
44.6±2.34abd2
Tris-based
Vapour (-70)
60
38.4±4.70abc3
Tris-based
-80
60
37.6±4.50abd3
Na Citrate
Vapour (-70)
60
37.0±4.36abc3
Na Citrate
-80
60
38.0±3.39abd3
Tris-based
Vapour (-70)
120
32.0±4.90abc4
Tris-based
-80
120
27.8±4.98abd4
Na Citrate
Vapour (-70)
120
27.6±5.08abc4
Na Citrate
-80
120
25.0±3.87abd4
Values within same column with different superscripts differ significantly (P < 0.05)
53 periods Mean±SE
Table 4.3 Mean (±SE) post-thaw sperm motility of SAMM ram semen frozen using
different extenders and freezing protocols at different incubation intervals following
thawing
Breed
SAMM
abcd1/2/3/4
Extender
Freezing
Incubation
periods
protocol (˚C)
after thawing (min)
Mean±SE
Tris-based
Vapour (-70)
0
52.8±3.22abc1
Tris-based
-80
0
54.6±3.92abd1
Na Citrate
Vapour (-70)
0
52.6±3.17abc1
Na Citrate
-80
0
52.6±3.17abd1
Tris-based
Vapour (-70)
30
50.0±3.16abc2
Tris-based
-80
30
46.6±4.07abd2
Na Citrate
Vapour (-70)
30
48.0±3.00abc2
Na Citrate
-80
30
46.0±3.67abd2
Tris-based
Vapour (-70)
60
37.2±4.40abc3
Tris-based
-80
60
29.0±4.58abd3
Na Citrate
Vapour (-70)
60
37.0±4.36abc3
Na Citrate
-80
60
31.0±5.79abd3
Tris-based
Vapour (-70)
120
30.0±4.18abc4
Tris-based
-80
120
20.0±0.01abd4
Na Citrate
Vapour (-70)
120
32.0±4.90abc4
Na Citrate
-80
120
22.0±1.22abd4
Values within same column with different superscripts differ significantly (P < 0.05)
4.3 Effect of breed, extender and freezing protocol on the in vitro performance of thawed
ram semen
The in vitro fertilization results are summarized in Table 4.4 and in Figure 4.1. All the extenders
and the freezing protocols recorded significant different (P<0.05) fertilization rate results
throughout the entire experiment, regardless of the breed. The breed recorded no significant
difference (P>0.05) and no interaction was found between the treatment groups. The mean
cleavage percentages recorded were 33.8±0.7% when Tris-egg-yolk based extender was used
with exposure to LN2 vapour prior to storage; 30.0 ± 0.7% when Tris-egg-yolk based extender
54 was used with freezing up to -80˚C (programmable freezer) prior to storage; 29.9 ± 0.7% when
Sodium Citrate-egg-yolk based extender was used with the aid of LN2 vapour prior to storage
and 25.7 ± 0.7% when Sodium Citrate-egg-yolk based extender was used with freezing up to 80˚C prior to storage.
Table 4.4 Mean (± SE) effect of extender and freezing protocol on in vitro fertilization of
ram semen
Extender
Freezing Protocol (˚C)
Tris-Egg-yolk based
Tris-Egg-yolk based
Sodium Citrate-Egg-yolk based
Sodium Citrate-Egg-yolk based
abcd
Cleavage (%)
33.82 ± 0.73a
Vapour (-70)
30.03 ± 0.73b
- 80
29.88 ± 0.73c
Vapour (-70)
25.71 ± 0.73d
- 80
Values within same column with different superscripts are significantly different (P<0.05)
35
30
25
20
SAMM
15
Dorper
10
5
0
Tris (‐70˚C)
Tris (‐80˚C)
Na (‐70˚C)
Na (‐80˚C)
Figure 4.1 Effect of breed on in vitro fertilization results of frozen-thawed ram semen
55 Where Tris (- 70˚C) is Tris-Egg-yolk based extender with a LN2 vapour (-70˚C) as a freezing
protocol, Tris (- 80˚C) is Tris-Egg-yolk based extender with freezing up to -80˚C, Na (-70˚C)
is Sodium Citrate-Egg-yolk based extender with a LN2 vapour (-70˚C) as a freezing protocol
and Na (-80˚C) is Sodium Citrate-Egg-yolk based extender with freezing up to -80˚C.
56 CHAPTER 5
DISCUSSION
5.1 Effect of breed (Dorper and SAMM) on fresh semen parameters prior to
cryopreservation
5.1.1 Semen volume
The breed of sheep has been hypothesized to be of great importance regarding the evaluation
of quality in fresh ram semen. The results of the current study are in agreement to the
hypothesis. The mean semen volume (1.4±0.1and 1.1±0.1 ml) for the Mutton Merino and
Dorper, respectively, suggests that the South African Mutton Merino produces significantly
higher semen volumes, compared to Dorper rams. A mean ejaculate volume of 1.2 ml has been
reported (Hamedani et al., 2013) in Dallagh rams and 1.1±0.1ml in goats (Batisda et al., 2009).
Similar significance differences for sheep breeds have been reported by other researchers. So
for example Mahoete (2010) reported significant differences in semen volume between Merino
and Sulu rams, while (Moghaddam et al., 2012) reported a significant difference between the
ArkharMerino × Ghezel and Ghezel × Baluchi rams. In addition the results are also in
agreement with values obtained from more recent studies. Malejane (2013) reported an
average semen volume of 1.1ml in Dorper rams, while Gil et al. (2003), documented ejaculate
volumes of 0.75 to 2 ml as being normal for rams when using artificial vagina for ram semen
collection. Hafez and Hafez (2000) also indicated a range of 0.5 to 2 ml in ejaculate volume
for mature rams and 0.5 to 0.7 ml in yearling rams. There are however number of factors that
can affect the volume of ram semen e.g scrotal circumference (Sarder, 2005; Devkota et al.,
2008; Hassan et al., 2009; Okere et al., 2011), age of the male, nutritional status, reproductive
management, method of semen collection, frequency of semen collection, season of the year
and responsiveness of the ram (Hafez and Hafez, 2000; Malejane, 2013).
57 5.1.2 Semen colour
As set out in table 4.1, there were no significant differences in semen colour between the
SAMM and Dorper sheep breeds. The general colour of the semen for both breeds was thin,
creamy. These findings fall within the range as reported by Ax et al. (2000), who documented
that fresh ram semen colour should vary from thin to thick creamy in consistency and, milkywhite or pale cream in colour. This parameter generally indicates the sperm density
(concentration), as well as possible contamination (Malejane, 2013). For the trial no
contamination was recorded throughout the entire experiment and thus the semen and the
technique of semen collection was considered to be of acceptable quality.
5.1.3 Sperm motility
Sperm motility is seen as a good indication of semen quality, fertilizing ability and animal
fertility in all species. Table 4.1 illustrates a high significant difference in sperm motility
(P<0.05) between the two breeds (81.3±0.1% and 70.2±0.2%) for the SAMM and Dorper males
respectively. This finding is in agreement with Karagiannidis et al. (2000), who reported a
range of 70 to 90% for sperm motility. Some factors have however been reported to affect
sperm motility and these include age (Brito et al., 2002; Salhab et al., 2003; Ghalban et al.,
2004; Kumar et al., 2010) and nutrition (Watanabe et al., 2003; Hamedani et al., 2013).
5.1.4 Sperm concentration
No significant differences were recorded between the two breeds. The results of this study are
in agreement with the findings of Gil et al. (2003), who reported a sperm concentration of
2.5×109 sperm/ml for rams to be normal and acceptable. This was also supported by Paulenz et
al., (2005) and O’Hara et al., (2010) who also documented acceptable sperm cell
concentrations of ≥2.5×109sperm/ml. According to the results of the present study, as set out
in Table 4.1, semen concentration of SAMM was higher than the semen concentration of
Dorper rams. Factors that could affect the sperm concentration include season, age of the male,
degree of stimulation and nutrition (Wolfenson et al., 2000; Brito et al., 2002; Salhab et al.,
2003; Zamiri et al, 2005; Kheradmand et al.,2006; Moghaddam et al., 2012).
58 5.1.5 Sperm viability
The percentage dead or live sperm was evaluated with the aid of the eosin/nigrosin stain,
whereby live sperm stain white and dead sperm stain red, as discussed in Chapter 3. This
staining method indicative of the live or dead status of the sperm cell, while also presenting a
good environment for the evaluation of the sperm cell morphology (Bjoerndahl et al., 2003).
The results of the current study regarding sperm viability are set out in Table 4.1. No significant
difference (P>0.05) was recorded between the two breeds. These findings (75.8± 1.9% and
71.2± 2.5%) fall within the range of 60 to 90% that was documented by Zamiri et al. (2010)
for the percentage live ram sperm but is higher than the findings of Schwalbach et al. (2006)
who reported live sperm percentage mean values of 64±3% and 70±2% for Dorper rams which
were managed intensively. This difference may be attributed to factors such as temperature and
season of semen collection (Leboeuf et al., 2000; Ghalban et al., 2004).
5.1.6 Sperm morphology
The data of the current trial indicate no significant difference in sperm morphology or
abnormalities between the SAMM and Dorper breeds (Table 4.1). These findings are in
agreement with other researchers (Hafez and Hafez, 2000; Karagiannidis et al., 2000). The
abnormalities recorded were relatively small (<10%) which is still acceptable as suggested by
most researchers. Within this range of less than 10% abnormalities, the majority of sperm were
seen with no tail, while some sperm were recorded with no head, no acrosome or a head with
a mid-piece but without a tail. Care must always be taken to prevent injury to the sperm cell
when making the smear. The relatively low percentage of abnormalities indicates that the
making of semen smears was acceptable.
5.2 Effects of breed difference, different extenders and different freezing protocols on the
viability of frozen-thawed ram semen
Fresh semen immediate after collection used in this research was found to be acceptable for
further processing, especially for dilution and freezing as in Table 4.1. Macroscopically, the
normal quality standards of fresh ram semen is 0.5 to 2.0 ml volume (Hafez and Hafez, 2000),
thin to thick creamy in consistency, milky to white or pale cream in colour (Ax et al., 2000).
When the microscope is used, the normal concentration ranges from 1500 million sperm/ml
(O’Hara et al., 2010), a sperm motility of 70 to 90% (Karagiannidis et al., 2000), a sperm
59 viability of 60 to 90% (Zamiri et al., 2010) and a percentage sperm abnormalities of ≤ 10%
(Karagiannidis et al., 2000).
The interaction of experimental factors in the current study recorded no significant differences
(P>0.05). However, taking a single factor into consideration, the incubation time intervals and
freezing protocols significantly affected the sperm motility (P<0.05). The negative relationship
that was recorded between time intervals and sperm motility post thawing thus prohibits the
use of cervical artificial insemination in ewes using frozen - thawed semen. These findings are
then in agreement with the findings of Gil et al. (2000). The low semen storage temperatures
used in the current study (-196˚C) is set to extend the fertile life span of the sperm cell, by
reducing the sperm metabolic rate. It has been documented by Hafez and Hafez, (2000) that as
metabolic rate increases the life span of the sperm cell decreases. This correlation has been
proven in the current study at temperatures of 39˚C following thawing. In addition, reports
concerning fresh semen indicated that the sperm has a short fertile life span outside the body
(Morrier et al., 2002) and this is due to increased cellular metabolism at higher temperatures.
Even though it was suggested by Brinsko et al. (2000) that the freezing of semen will increase
its longevity, the biggest challenge is fertility of the sperm cell after thawing. Reduction in the
fertilization capacity has typically been attributed to a reduced rate in sperm motility and the
freeze-thaw-induced morphological and genomic abnormalities (Morris et al., 2002; Martin et
al., 2004). The semen frozen following the use of liquid nitrogen vapour prior to storage
recorded a better sperm motility than semen frozen at -80˚C prior to storage in liquid nitrogen.
It is thus suggested that in programmable semen freezing programs, semen should be exposed
to liquid nitrogen vapour immediately prior to the storage into liquid nitrogen (-196˚C) and
should not be frozen at -80˚C. According to the knowledge gained thus far, there is no published
information regarding programmable freezing of the ram semen using temperatures similar to
those used in this study. The use of the programmable freezer has however been reported to
produce better sperm motility (Hammadeh et al., 2001; Clulow et al., 2008), with less
cryodamage to the sperm cell (Petyim and Choavaratana, 2006).
The use of a Tris-egg yolk based extender showed better post thawing motility than the Na
Citric-egg yolk based extender in both breeds of sheep. Similar findings have been reported
where semen was stored at 5˚C and 20˚C (Paulenz et al., 2002). Both extenders used in this
study have been recommended by other researchers to be acceptable for ram semen
cryopreservation in programs of genetic animal material improvement. In addition, Tris-egg
yolk based extender has been documented to provide adequate cryoprotection (Salamon and
60 Maxwell, 2000). However, the difference in performance may be due to the types and
concentrations of sugars used. So for example, with an increase in sugar concentrations, all
post-thawed sperm characteristics were generally improved and higher sperm motility has been
reported with the use of trehalose (Jafaroghli et al., 2011). Furthermore, trehalose has been
reported to have certain antioxidant properties (Aisen et al., 2002), compared to glucose used
in this study. The egg yolk has been stated to make semen evaluation problems (Moussa et al.,
2002), increase the risk of microbial contamination and thereby allow the production of
endotoxin, hence lowering the motility of the sperm (Aires et al., 2003).
5.3 Effect of breed, extender and freezing protocol on the in vitro performance of thawed
ram semen
The sperm parameters of the frozen-thawed semen showed no significant difference (P>0.05)
between the two breeds. The extenders and the freezing protocols were however significantly
(P< 0.05) different throughout the entire experiment, regardless of the breed. These results are
contradictory with the results of other researchers (Mahoete, 2010). The standard error of the
present work (0.73) seems to be very small as compared to the standard error (3.7) of results
reported by Mahoete (2010) in the Merino breed semen. This larger variation could be
attributed to different factors, however, it is difficult to interpret the cause of this difference, as
the materials and methods used were different. The use of a Tris-egg yolk based extender
reported better cleavage results (31.91± 0.73%), compared to use of a sodium citrate-egg yolk
based extender (27.8 ± 0.73%). When smaller percentages of egg yolk (10%) were included,
the sodium citrate-egg yolk based extender exhibited better cleavage (Mahoete, 2010), while
in this study, 20% egg yolk was used. Thus, further research is warranted on the effective levels
of egg yolk inclusion in the sodium citrate extender. These in vitro results have confirmed the
results of the current experiment for Tris-egg yolk based extender vs sodium citrate-egg yolk
based extender and are in agreement with the work of Valente et al. (2009). Although the eggyolk used was less (15%) (Valente et al., 2009) while the optimal level is allowed to be 20%
(Forouzanfar et al., 2010), which was used in the current study. It is further documented that
the freezing of ram semen in a Tris-egg yolk base with 20% egg yolk, offers a higher number
of viable spermatozoa that can be used during IVF and Artificial Insemination (Forouzanfar et
al., 2010). Furthermore, the results of the current study are in agreement with results of GarcíaÁlvarez et al. (2009a) who reported a range of 31% to 59% cleavage for in vitro results, when
61 vapour was used prior to storage in to liquid nitrogen. Compared to Dorado et al. (2007) who
reported that Tris-based extender results in better IVF results, compared to other extenders.
When liquid nitrogen vapour was used in the current study, better in vitro results were obtained,
compared to the use of -80˚C, prior to storage. No relevant literature could be found for the
freezing of up to -80˚C, prior to storage in liquid nitrogen.
Variations in cleavage percentages may be due to the methods of semen quality control applied
and quality assurance procedures and contributing conditions in the embryology laboratory. In
the present work, the selection of oocytes for in vitro fertilization was performed with the aid
of the brilliant cresyl blue technique, to ensure that only oocytes of good quality were used
(Bhojwani et al., 2007). The experience of the researcher has also been reported to contribute
to variation in sperm quality results (Malejane, 2013), thus affecting in vitro results. Different
methods of semen collection may also have contributed to the variations of in vitro fertilization
results experienced (García-Álvarez et al., 2009b). However, the semen collection methods
used in this study was reported to provide better results compared to other collection methods
(Malejane, 2013). The reliable and repeatable ejaculates obtained are an indication of an
acceptable collection technique (artificial vagina) used.
The results thus suggest that frozen-thawed ram semen can perform well in in vitro fertilization
procedures, if a Tris-egg yolk based extender is used as the diluent in combination with
exposure to liquid nitrogen vapour as a freezing protocol prior to storage. Further work is
warranted to confirm these results in vivo.
62 CHAPTER 6
GENERAL CONCLUSIONS
The semen volume and sperm motility parameters showed a significant difference between the
two sheep breeds (P<0.05) where the South African Mutton Merino (SAMM) semen performed
better than the Dorper semen for the entire period of the trial. There was however no significant
differences for the breeds in terms of semen colour, sperm concentration, percentage live and
dead sperm and percentage normal sperm. Based on these results it could be concluded that
further work must be done with a larger number of animals to increase the statistical precision
and significance. This is due to the fact that few numbers of rams per breed were used in the
current study and the fact that a difference was found only between two of the semen
parameters.
The semen frozen using liquid nitrogen vapour prior to storage exhibited better sperm motility
than semen frozen to -80˚C (programmable freezer) prior to storage. The sperm viability of the
frozen ram semen was inversely proportional with the incubation time increase after thawing.
Only the freezing protocols and the time intervals showed a significant difference (P<0.05)
throughout the entire experiment. Statistically, breed difference and the different semen
extenders had no significant effect (P>0.05). However, when Tukey’s grouping was used
(studentised range), the Tris-egg yolk based extender exhibited a better performance at the
beginning of the experiment, compared to the Sodium Citrate-egg yolk based extender. It could
thus be concluded that the Tris-egg yolk based extender should be used as a diluent for ram
semen cryopreservation and freezing with the aid of liquid nitrogen vapour, prior to storage.
This will lead to a better fertility if insemination is done immediately after thawing, or
alternatively the laparoscopic method will remain an alternative.
The semen frozen by the use of liquid nitrogen vapour prior to storage, showed better oocyte
cleavage rates post in vitro fertilization, compared to the semen frozen to -80˚C (programmable
freezer), prior to storage. Only the freezing protocols and semen extenders exhibited a
significant difference on oocyte cleavage rate post in vitro fertilization throughout the entire
experiment. Statistically the different breeds showed no significant difference in cleavage rate
post in vitro fertilization. Therefore, it could be concluded that in general frozen-thawed ram
63 semen (irrespective of the breed) can perform well in the in vitro fertilization procedures used
if the Tris-egg-yolk based extender is used as a diluent in combination with exposure to liquid
nitrogen vapour prior to storage as a freezing protocol. Further research following thawing is
warranted to confirm fertility results in vivo. The results thus also lead to the conclusion that
any of the two breeds can be used for the in vitro fertilization programs.
RECOMMENDATIONS
It is recommended that further research must be performed with a larger number of animals for
better statistical precision or significance to determine the effect of breed on the quality of ram
semen. The Tris-egg-yolk based extender should be used as diluent, in combination with
exposure to liquid nitrogen vapour as a freezing protocol prior to storage. More research is also
warranted to evaluate the results of these cryopreservation techniques in the field of in vivo
fertility.
64 ABSTRACT
EVALUATION OF CRYOPRESERVED RAM SEMEN
FOLLOWING FERTILIZATION IN VITRO
by
Masilo Henoke Mohlomi
Supervisor: Mr. M. B. Raito
Co-Supervisors: Prof. J.P.C. Greyling
Dr. A.M. Jooste
Department of Animal, Wildlife and Grassland Sciences
University of the Free State
Bloemfontein
Degree: M.Sc. (Agric.)
Three experiments were done at the University of Free State in Bloemfontain in 2014 and in
2015. Experiment 1 was carried to evaluate the effect of breed (Dorper and SAMM) on fresh
ram semen parameters before cryopreservation. Experiment 2 was carried to evaluate the
effects of breed difference, different extenders and different freezing protocols on the viability
of frozen ram semen during different incubation periods after thawing. Experiment 3 was
carried to evaluate effects of breed, extender and freezing protocol on in vitro fertilizing
65 capacity of frozen-thawed ram semen. Experiments 1 and 2 were done in 2014 during the
breeding season while experiment 2 was done in 2015 from mid of January until the end of
April.
For the first trial, the semen was collected with the aid of artificial vagina from two breeds of
rams. The ejaculates were evaluated for the following parameters: semen volume, semen
colour, sperm motility, sperm concentration, percentage live/dead sperm and sperm
morphology. The parameters were then compared against the breeds to assess the breed effect.
The semen volume and sperm motility showed a significant difference between the two breeds
(P<0.05) in that South African Mutton Merino (SAMM) semen recorded better motility and
higher semen volume than Dorper semen for the entire experiment. There was no significant
difference between breeds for semen colour, sperm concentration, percentage live and dead
sperm and sperm morphology.
For the second trial, the study to evaluate the effect of different breeds, extenders and freezing
protocols on the sperm motility of ram semen including survival at different incubation periods
after thawing was conducted during the natural breeding season. Semen was collected from
two breeds of rams (South African Marten Merino (SAMM) and Dorper). After fresh semen
evaluation and selection of good quality ejaculates, semen was pooled for each breed and
divided into two then randomly allocated to Tris-egg yolk and Na-Citrate egg yolk based
extenders. Semen samples were further divided into two more groups and allocated randomly
to two different freezing protocols (freezing up to -20˚C using the programmable freezer and
then by liquid nitrogen vapour prior to storage vs freezing to -80˚C prior to storage).
Microscopic evaluation of the semen for sperm motility was performed 24 hrs following
freezing. The semen frozen by the use of liquid nitrogen vapour prior to storage recorded better
sperm motility than semen frozen to -80˚C prior to storage. The viability of the frozen ram
semen was inversely proportional with incubation time increase after thawing. Only the
freezing protocols and incubation periods exhibited a significant difference in sperm motility
throughout the experiment. Statistically, the difference breeds and the different semen
extenders recorded no significant difference. However, when Tukey’s grouping was used
(studentised range), the Tris-egg yolk based extender showed better sperm motility compared
to the Na-Citrate egg yolk based extender. The results suggest that the breed does not have
impact on semen freezing and the Tris-egg yolk based extender is better suited as a diluent for
ram semen cryopreservation. Results also indicate that freezing with the aid of liquid nitrogen
66 vapour prior to storage will lead to a better fertility if insemination is done immediately after
thawing or alternatively the laparoscopic method will remain an alternative.
In the last experiment, ovaries were collected during spring and at the beginning of summer at
the Bloemfontein abattoir from unknown, untreated sheep and transported to the laboratory in
sterile saline water (37˚C) in the flask. Cumulus oocytes complexes (COC’s) were recovered
from the ovarian follicles by aspiration method, washed and stained in brilliant crysel blue for
selection of good quality oocytes. The COC were then washed and matured in vitro for 24 hrs
before in vitro fertilization. The frozen semen (frozen by different extenders and different
freezing protocols and from different breeds) was thawed and then centrifuged twice at 1500
rpm for 8minutes at 38˚C. Then 50 µl of the prepared semen was placed in the IVF aliquot
containing the mature oocytes and incubated for 18 hrs at 38˚C (5% carbon dioxide and 90%
relative humidity). After 18 hrs of oocyte-sperm incubation, presumptive zygotes were
removed from the IVF aliquots into a 1.5 ml eppendorf tube containing 100 µl of pre-incubated
M199 + FBS and vortexed for 1.5 min to strip off the cumulus cells. After vortexing, zygotes
were washed 3 times in both M199 + FBS and pre-incubated culture media (SOF-BSA)
droplets after which the zygotes were allocated to groups of 20 per aliquot which were covered
with mineral oil and placed in gas chamber with 3 gases, then incubated at 39˚C for 7 days in
humidified incubator (5% oxygen, 5% carbon dioxide and 90% nitrogen). During this
incubation time, the culture medium was changed at 48 hrs after fertilization to SOF-FBS by
aspirating medium from the aliquots and replacing the medium with the fresh pre-incubated
medium with the aid of a pipette, during which the cleavage rate (2 to 8 cells) was examined
and embryos were then separated according to number of cell division. The media was changed
again during day 5. Microscopic evaluations were performed during day 6, and day 7 for the
development into morula and blastocyst respectively and results were recorded into the excel
sheet for data analysis.
The semen frozen by with the aid of liquid nitrogen vapour prior to storage recorded better
cleavage compared to semen frozen to -80˚C prior to storage. Only the freezing protocols and
semen extenders exhibited a significant difference in post in vitro fertilization throughout the
entire in vitro fertilization experiment. Statistically the different breeds had no significant
difference in post in vitro fertilization. The results suggests that, in general frozen-thawed ram
semen irrespective of the breed can perform well in the in vitro fertilization procedures used if
the Tris-egg-yolk based extender is used as diluents in combination with exposure to liquid
nitrogen vapour prior to storage as a freezing protocol. Further research following thawing is
67 warranted to confirm fertility results in vivo. The results thus also indicate that the breed has
no effect on the in vitro results of frozen-thawed ram semen.
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